Oscillatory behavior of neutrophils under opposing chemoattractant gradients supports a winner-take-all mechanism.
ABSTRACT: Neutrophils constitute the largest class of white blood cells and are the first responders in the innate immune response. They are able to sense and migrate up concentration gradients of chemoattractants in search of primary sites of infection and inflammation through a process known as chemotaxis. These chemoattractants include formylated peptides and various chemokines. While much is known about chemotaxis to individual chemoattractants, far less is known about chemotaxis towards many. Previous studies have shown that in opposing gradients of intermediate chemoattractants (interleukin-8 and leukotriene B4), neutrophils preferentially migrate toward the more distant source. In this work, we investigated neutrophil chemotaxis in opposing gradients of chemoattractants using a microfluidic platform. We found that primary neutrophils exhibit oscillatory motion in opposing gradients of intermediate chemoattractants. To understand this behavior, we constructed a mathematical model of neutrophil chemotaxis. Our results suggest that sensory adaptation alone cannot explain the observed oscillatory motion. Rather, our model suggests that neutrophils employ a winner-take-all mechanism that enables them to transiently lock onto sensed targets and continuously switch between the intermediate attractant sources as they are encountered. These findings uncover a previously unseen behavior of neutrophils in opposing gradients of chemoattractants that will further aid in our understanding of neutrophil chemotaxis and the innate immune response. In addition, we propose a winner-take-all mechanism allows the cells to avoid stagnation near local chemical maxima when migrating through a network of chemoattractant sources.
Project description:The dynamics of neutrophil chemotaxis under competing chemoattractant gradients was studied using a microfluidic platform. This microfluidic platform, which establishes a stable and dynamic gradient of chemoattractants across a cell culture chamber, enabled the investigation of human neutrophil migration patterns in the presences of four different chemoattractants (leukotriene B(4), chemokine C-X-C motif ligands 2 and 8, and fMLP) and competing gradients of all pairwise combinations. The migration patterns for individual cells were tracked and quantitatively analyzed, and the results suggest a hierarchy among these chemoattractants of fMLP > CXCL8 > CXCL2 > leukotriene B(4). In all conditions, over 60% of neutrophils exposed to a competing gradient move toward the stronger signal though the weaker chemoattractant still influences neutrophil motility. These results yield insight about how each chemoattractant contributes to overall neutrophil chemotaxis within complex physiological environments.
Project description:Neutrophils are the first responders to infection and play a pivotal role in many inflammatory diseases, including sepsis. Recent studies have shown that lipopolysaccharide (LPS), a classical pattern recognition molecule, dynamically programs innate immune responses. In this study, we show that pre-treatment with super-low levels of LPS [1 ng/mL] significantly dysregulate neutrophil migratory phenotypes, including spontaneous migration and altering neutrophil decision-making. To quantify neutrophil migratory decision-making with single-cell resolution, we developed a novel microfluidic competitive chemotaxis-chip (μC3) that exposes cells in a central channel to competing chemoattractant gradients. In this reductionist approach, we use two chemoattractants: a pro-resolution (N-Formyl-Met-Leu-Phe, fMLP) and pro-inflammatory (Leukotriene B4, LTB4) chemoattractant to model how a neutrophil makes a decision to move toward an end target chemoattractant (e.g., bacterial infection) vs. an intermediary chemoattractant (e.g., inflammatory signal). We demonstrate that naïve neutrophils migrate toward the primary end target signal in higher percentages than toward the secondary intermediary signal. As expected, we found that training with high dose LPS [100 ng/mL] influences a higher percentage of neutrophils to migrate toward the end target signal, while reducing the percentage of neutrophils that migrate toward the intermediary signal. Surprisingly, super-low dose LPS [1 ng/mL] significantly changes the ratios of migrating cells and an increased percentage of cells migrate toward the intermediary signal. Significantly, there was also an increase in the numbers of spontaneously migrating neutrophils after treatment with super-low dose LPS. These results shed light onto the directional migratory decision-making of neutrophils exposed to inflammatory training signals. Understanding these mechanisms may lead to the development of pro-resolution therapies that correct the neutrophil compass and reduce off-target organ damage.
Project description:Neutrophils and Dictyostelium use conserved signal transduction pathways to decipher chemoattractant gradients and migrate directionally. In both cell types, addition of chemoattractants stimulates the production of cAMP, which has been suggested to regulate chemotaxis. We set out to define the mechanism by which chemoattractants increase cAMP levels in human neutrophils. We show that chemoattractants elicit a rapid and transient activation of adenylyl cyclase (AC). This activation is sensitive to pertussis toxin treatment but independent of phosphoinositide-3 kinase activity and an intact cytoskeleton. Remarkably, and in sharp contrast to Galpha(s)-mediated activation, chemoattractant-induced AC activation is lost in cell lysates. Of the nine, differentially regulated transmembrane AC isoforms in the human genome, we find that isoforms III, IV, VII, and IX are expressed in human neutrophils. We conclude that the signal transduction cascade used by chemoattractants to activate AC is conserved in Dictyostelium and human neutrophils and is markedly different from the canonical Galpha(s)-meditated pathway.
Project description:Neutrophil recruitment and directional movement toward chemotactic stimuli are important processes in innate immune responses. This study examines the role of Fer kinase in neutrophil recruitment and chemotaxis to various chemoattractants in vitro and in vivo. Mice targeted with a kinase-inactivating mutation (Fer(DR/DR)) or wild type (WT) were studied using time-lapse intravital microscopy to examine leukocyte recruitment and chemotaxis in vivo. In response to keratinocyte-derived cytokine, no difference in leukocyte chemotaxis was observed between WT and Fer(DR/DR) mice. However, in response to the chemotactic peptide WKYMVm, a selective agonist of the formyl peptide receptor, a 2-fold increase in leukocyte emigration was noted in Fer(DR/DR) mice (p < 0.05). To determine whether these defects were due to Fer signaling in the endothelium or other nonhematopoietic cells, bone marrow chimeras were generated. WKYMVm-induced leukocyte recruitment in chimeric mice (WT bone marrow to Fer(DR/DR) recipients or vice versa) was similar to WT mice, suggesting that Fer kinase signaling in both leukocytes and endothelial cells serves to limit chemotaxis. Purified Fer(DR/DR) neutrophils demonstrated enhanced chemotaxis toward end target chemoattractants (WKYMVm and C5a) compared with WT using an under-agarose gel chemotaxis assay. These defects were not observed in response to intermediate chemoattractants (keratinocyte-derived cytokine, MIP-2, or LTB(4)). Increased WKYMVm-induced chemotaxis of Fer(DR/DR) neutrophils correlated with sustained PI3K activity and reduced reliance on the p38 MAPK pathway compared with WT neutrophils. Together, these data identify Fer as a novel inhibitory kinase for neutrophil chemotaxis toward end target chemoattractants through modulation of PI3K activity.
Project description:Neutrophils are key cellular components of the innate immune response and characteristically migrate from the blood towards and throughout tissues. Their migratory process is complex, guided by multiple chemoattractants released from injured tissues and microbes. How neutrophils integrate the various signals in the tissue microenvironment and mount effective responses is not fully understood. Here, we employed microfluidic mazes that replicate features of interstitial spaces and chemoattractant gradients within tissues to analyze the migration patterns of human neutrophils. We find that neutrophils respond to LTB4 and fMLF gradients with highly directional migration patterns and converge towards the source of chemoattractant. We named this directed migration pattern convergent. Moreover, neutrophils respond to gradients of C5a and IL-8 with a low-directionality migration pattern and disperse within mazes. We named this alternative migration pattern divergent. Inhibitors of MAP kinase and PI-3 kinase signaling pathways do not alter either convergent or divergent migration patterns, but reduce the number of responding neutrophils. Overlapping gradients of chemoattractants conserve the convergent and divergent migration patterns corresponding to each chemoattractant and have additive effects on the number of neutrophils migrating. These results suggest that convergent and divergent neutrophil migration-patterns are the result of simultaneous activation of multiple signaling pathways.
Project description:Neutrophils act as the first line of defence in the human immune system by migrating to the site of abnormal events and performing their designated roles. One major signalling pathway that drives neutrophil action in vivo is the p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Herein, a microfluidic platform is employed to explore the mechanistic role of p38 MAPK in neutrophil chemotaxis. Neutrophils, with and without p38 MAPK inhibition, were exposed to pairwise competing gradients of chemotaxis-inducing molecules. Overall, p38 MAPK inhibitor-treated neutrophils were still capable of moving toward a chemoattractant signal; however, the hierarchy of neutrophil response to various chemoattractants changed and there was more deviation from direct movement toward a chemoattractant signal in p38 MAPK-blocked cells. In a parallel fluorescence imaging study, neutrophil expression of surface receptors (CXCR1, FPR2, BLTR, CD11b and CD66b) changed when comparing untreated and p38 MAPK-blocked cells. All results demonstrate that the p38 MAPK-dependent pathway plays a critical role in neutrophil chemotaxis and this role is, in part, through the regulation of surface receptor expression. These data regarding how receptor expression and chemotaxis are influenced by the p38 MAPK pathways lend insight into neutrophil behaviour in physiological environments and the potential manipulation of p38 MAPK for therapeutic purposes.
Project description:Chemotaxis is the ability to migrate towards the source of chemical gradients. It underlies the ability of neutrophils and other immune cells to hone in on their targets and defend against invading pathogens. Given the importance of neutrophil migration to health and disease, it is crucial to understand the basic mechanisms controlling chemotaxis so that strategies can be developed to modulate cell migration in clinical settings. Because of the complexity of human genetics, Dictyostelium and HL60 cells have long served as models system for studying chemotaxis. Since many of our current insights into chemotaxis have been gained from these two model systems, we decided to compare them side by side in a set of winner-take-all races, the Dicty World Races. These worldwide competitions challenge researchers to genetically engineer and pharmacologically enhance the model systems to compete in microfluidic racecourses. These races bring together technological innovations in genetic engineering and precision measurement of cell motility. Fourteen teams participated in the inaugural Dicty World Race 2014 and contributed cell lines, which they tuned for enhanced speed and chemotactic accuracy. The race enabled large-scale analyses of chemotaxis in complex environments and revealed an intriguing balance of speed and accuracy of the model cell lines. The successes of the first race validated the concept of using fun-spirited competition to gain insights into the complex mechanisms controlling chemotaxis, while the challenges of the first race will guide further technological development and planning of future events.
Project description:Neutrophils, in response to a chemoattractant gradient, undergo dynamic F-actin remodeling, a process important for their directional migration or chemotaxis. However, signaling mechanisms for chemoattractants to regulate the process are incompletely understood. Here, we characterized chemoattractant-activated signaling mechanisms that regulate cofilin dephosphorylation and actin cytoskeleton reorganization and are critical for neutrophil polarization and chemotaxis. In neutrophils, chemoattractants induced phosphorylation and inhibition of GSK3 via both PLC?-PKC and PI3K?-AKT pathways, leading to the attenuation of GSK3-mediated phosphorylation and inhibition of the cofilin phosphatase slingshot2 and an increase in dephosphorylated, active cofilin. The relative contribution of this GSK3-mediated pathway to neutrophil chemotaxis regulation depended on neutrophil polarity preset by integrin-induced polarization of PIP5K1C. Therefore, our study characterizes a signaling mechanism for chemoattractant-induced actin cytoskeleton remodeling and elucidates its context-dependent role in regulating neutrophil polarization and chemotaxis.
Project description:Chemotaxis is a fundamental biological process where complex chemotactic gradients are integrated and prioritized to guide cell migration toward specific locations. To understand the mechanisms of gradient dependent cell migration, it is important to develop in vitro models that recapitulate key attributes of the chemotactic cues present in vivo. Current in vitro tools for studying cell migration are not amenable to easily study the response of neutrophils to dual gradients. Many of these systems require external pumps and complex setups to establish and maintain the gradients. Here we report a simple yet innovative microfluidic device for studying cell migration in the presence of dual chemotactic gradients through a 3-dimensional substrate. The device is tested and validated by studying the migration of the neutrophil-like cell line PLB-985 to gradients of fMLP. Furthermore, the device is expanded and used with heparinised whole blood, whereupon neutrophils were observed to migrate from whole blood towards gradients of fMLP eliminating the need for any neutrophil purification or capture steps.
Project description:Neutrophil recruitment guided by chemotactic cues is a central event in host defense against infection and tissue injury. While the mechanisms underlying neutrophil chemotaxis have been extensively studied, these are just recently being addressed by using high-content approaches or surface-bound chemotactic gradients (haptotaxis) in vitro. Here, we report a haptotaxis assay, based on the classic under-agarose assay, which combines an optical patterning technique to generate surface-bound formyl peptide gradients as well as an automated imaging and analysis of a large number of migration trajectories. We show that human neutrophils migrate on covalently-bound formyl-peptide gradients, which influence the speed and frequency of neutrophil penetration under the agarose. Analysis revealed that neutrophils migrating on surface-bound patterns accumulate in the region of the highest peptide concentration, thereby mimicking in vivo events. We propose the use of a chemotactic precision index, gyration tensors and neutrophil penetration rate for characterizing haptotaxis. This high-content assay provides a simple approach that can be applied for studying molecular mechanisms underlying haptotaxis on user-defined gradient shape.