Collagen concentration regulates neutrophil extravasation and migration in response to infection in an endothelium dependent manner.

Introduction: As the body's first line of defense against disease and infection, neutrophils must efficiently navigate to sites of inflammation; however, neutrophil dysregulation contributes to the pathogenesis of numerous diseases that leave people susceptible to infections. Many of these diseases are also associated with changes to the protein composition of the extracellular matrix. While it is known that neutrophils and endothelial cells, which play a key role in neutrophil activation, are sensitive to the mechanical and structural properties of the extracellular matrix, our understanding of how protein composition in the matrix affects the neutrophil response to infection is incomplete. Methods: To investigate the effects of extracellular matrix composition on the neutrophil response to infection, we used an infection-on-a-chip microfluidic device that replicates a portion of a blood vessel endothelium surrounded by a model extracellular matrix. Model blood vessels were fabricated by seeding human umbilical vein endothelial cells on 2, 4, or 6 mg/mL type I collagen hydrogels. Primary human neutrophils were loaded into the endothelial lumens and stimulated by adding the bacterial pathogen Pseudomonas aeruginosa to the surrounding matrix. Results: Collagen concentration did not affect the cell density or barrier function of the endothelial lumens. Upon infectious challenge, we found greater neutrophil extravasation into the 4 mg/mL collagen gels compared to the 6 mg/mL collagen gels. We further found that extravasated neutrophils had the highest migration speed and distance in 2mg/mL gels and that these values decreased with increasing collagen concentration. However, these phenomena were not observed in the absence of an endothelial lumen. Lastly, no differences in the percent of extravasated neutrophils producing reactive oxygen species were observed across the various collagen concentrations. Discussion: Our study suggests that neutrophil extravasation and migration in response to an infectious challenge are regulated by collagen concentration in an endothelial cell-dependent manner. The results demonstrate how the mechanical and structural aspects of the tissue microenvironment affect the neutrophil response to infection. Additionally, these findings underscore the importance of developing and using microphysiological systems for studying the regulatory factors that govern the neutrophil response.

Diverse bacteria elicit distinct neutrophil responses in a physiologically relevant model of infection.

An efficient neutrophil response is critical for fighting bacterial infections, which remain a significant global health concern; therefore, modulating neutrophil function could be an effective therapeutic approach. While we have a general understanding of how neutrophils respond to bacteria, how neutrophil function differs in response to diverse bacterial infections remains unclear. Here, we use a microfluidic infection-on-a-chip device to investigate the neutrophil response to four bacterial species: Pseudomonas aeruginosa, Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus. We find enhanced neutrophil extravasation to L. monocytogenes, a limited overall response to S. aureus, and identify IL-6 as universally important for neutrophil extravasation. Furthermore, we demonstrate a higher percentage of neutrophils generate reactive oxygen species (ROS) when combating gram-negative bacteria versus gram-positive bacteria. For all bacterial species, we found the percentage of neutrophils producing ROS increased following extravasation through an endothelium, underscoring the importance of studying neutrophil function in physiologically relevant models.

Centriole and Golgi microtubule nucleation are dispensable for the migration of human neutrophil-like cells.

Neutrophils migrate in response to chemoattractants to mediate host defense. Chemoattractants drive rapid intracellular cytoskeletal rearrangements including the radiation of microtubules from the microtubule-organizing center (MTOC) toward the rear of polarized neutrophils. Microtubules regulate neutrophil polarity and motility, but little is known about the specific role of MTOCs. To characterize the role of MTOCs on neutrophil motility, we depleted centrioles in a well-established neutrophil-like cell line. Surprisingly, both chemical and genetic centriole depletion increased neutrophil speed and chemotactic motility, suggesting an inhibitory role for centrioles during directed migration. We also found that depletion of both centrioles and GM130-mediated Golgi microtubule nucleation did not impair neutrophil directed migration. Taken together, our findings demonstrate an inhibitory role for centrioles and a resilient MTOC system in motile human neutrophil-like cells.

Microphysiological Systems for Studying Cellular Crosstalk During the Neutrophil Response to Infection.

Neutrophils are the primary responders to infection, rapidly migrating to sites of inflammation and clearing pathogens through a variety of antimicrobial functions. This response is controlled by a complex network of signals produced by vascular cells, tissue resident cells, other immune cells, and the pathogen itself. Despite significant efforts to understand how these signals are integrated into the neutrophil response, we still do not have a complete picture of the mechanisms regulating this process. This is in part due to the inherent disadvantages of the most-used experimental systems: in vitro systems lack the complexity of the tissue microenvironment and animal models do not accurately capture the human immune response. Advanced microfluidic devices incorporating relevant tissue architectures, cell-cell interactions, and live pathogen sources have been developed to overcome these challenges. In this review, we will discuss the in vitro models currently being used to study the neutrophil response to infection, specifically in the context of cell-cell interactions, and provide an overview of their findings. We will also provide recommendations for the future direction of the field and what important aspects of the infectious microenvironment are missing from the current models.

Immune Cell Paracrine Signaling Drives the Neutrophil Response to A. fumigatus in an Infection-on-a-Chip Model.

INTRODUCTION: Neutrophils act as first responders during an infection, following signals from the pathogen as well as other host cells to migrate from blood vessels to the site of infection. This tightly regulated process is critical for pathogen clearance and, in many cases, eliminates the pathogen without the need for an additional immune response. It is, therefore, critical to understand what signals drive neutrophil migration to infection in a physiologically relevant environment. METHODS: In this study, we used an infection-on-a-chip model to recapitulate many important aspects of the infectious microenvironment including an endothelial blood vessel, an extracellular matrix, and the environmental fungal pathogen Aspergillus fumigatus. We then used this model to visualize the innate immune response to fungal infection. RESULTS: We found that A. fumigatus germination dynamics are influenced by the presence of an endothelial lumen. Furthermore, we demonstrated that neutrophils are recruited to and swarm around A. fumigatus hyphae and that the presence of monocytes significantly increases the neutrophil response to A. fumigatus. Using secreted protein analysis and blocking antibodies, we found that this increased migration is likely due to signaling by MIP-1 family proteins. Finally, we demonstrated that signal relay between neutrophils, mediated by LTB4 signaling, is also important for sustained neutrophil migration and swarming in response to A. fumigatus infection in our system. CONCLUSIONS: Taken together, these results suggest that paracrine signaling from both monocytes and neutrophils plays an important role in driving the neutrophil response to A. fumigatus.

A reconfigurable microscale assay enables insights into cancer-associated fibroblast modulation of immune cell recruitment.

Innate immune cell infiltration into neoplastic tissue is the first line of defense against cancer and can play a deterministic role in tumor progression. Here, we describe a series of assays, using a reconfigurable microscale assay platform (i.e. Stacks), which allows the study of immune cell infiltration in vitro with spatiotemporal manipulations. We assembled Stacks assays to investigate tumor-monocyte interactions, re-education of activated macrophages, and neutrophil infiltration. For the first time in vitro, the Stacks infiltration assays reveal that primary tumor-associated fibroblasts from specific patients differ from that associated with the benign region of the prostate in their ability to limit neutrophil infiltration as well as facilitate monocyte adhesion and anti-inflammatory monocyte polarization. These results show that fibroblasts play a regulatory role in immune cell infiltration and that Stacks has the potential to predict individual patients' cancer-immune response.

Neutrophil trafficking on-a-chip: an in vitro, organotypic model for investigating neutrophil priming, extravasation, and migration with spatiotemporal control.

Neutrophil trafficking is essential for a strong and productive immune response to infection and injury. During acute inflammation, signals from resident immune cells, fibroblasts, and the endothelium help to prime, attract, and activate circulating neutrophils at sites of inflammation. Due to current limitations with in vitro and animal models, our understanding of these events is incomplete. In this paper, we describe a microfluidic technology which incorporates a lumen-based vascular component with a high degree of spatiotemporal control to facilitate the study of neutrophil trafficking using primary human cells. The improved spatiotemporal control allows functional selection of neutrophils based on their migratory capacity. We use this technology to investigate neutrophil-endothelial interactions and find that these interactions are necessary for robust neutrophil chemotaxis to interleukin-8 (IL-8) and priming of the neutrophils. In agreement with previous studies, we observed that transendothelial migration (TEM) is required for neutrophils to enter a primed phenotypic state. TEM neutrophils not only produce a significantly higher amount of reactive oxygen species (ROS) when treated with PMA, but also upregulate genes involved in ROS production (CYBB, NCF1, NFKB1, NFKBIA), cell adhesion (CEACAM-8, ITGAM), and chemokine receptors (CXCR2, TNFRSF1A). These results suggest that neutrophil-endothelial interactions are crucial to neutrophil chemotaxis and ROS generation.

Neutrophil plasticity in the tumor microenvironment.

Neutrophils act as the body's first line of defense against infection and respond to diverse inflammatory cues, including cancer. Neutrophils display plasticity, with the ability to adapt their function in different inflammatory contexts. In the tumor microenvironment, neutrophils have varied functions and have been classified using different terms, including N1/N2 neutrophils, tumor-associated neutrophils, and polymorphonuclear neutrophil myeloid-derived suppressor cells (PMN-MDSCs). These populations of neutrophils are primarily defined by their functional phenotype, because few specific cell surface markers have been identified. In this review, we will discuss neutrophil polarization and plasticity and the function of proinflammatory/anti-inflammatory and protumor/antitumor neutrophils in the tumor microenvironment. We will also discuss how neutrophils with the ability to suppress T-cell activation, referred to by some as PMN-MDSCs, fit into this paradigm.

Neutrophil Reverse Migration and a Chemokinetic Resolution.

Neutrophil migration to sites of injury or infection is a critical first step in innate immunity. Resolution of neutrophil inflammation is essential for tissue repair but is less well understood. Recent work using intravital imaging in mice to visualize neutrophil dynamics at sterile injuries has advanced our understanding of neutrophil reverse migration and resolution of inflammation in mammals.

Interaction with an endothelial lumen increases neutrophil lifetime and motility in response to P aeruginosa.

Neutrophil infiltration into tissues is essential for host defense and pathogen clearance. Although many of the signaling pathways involved in the transendothelial migration of neutrophils are known, the role of the endothelium in regulating neutrophil behavior in response to infection within interstitial tissues remains unclear. Here we developed a microscale 3-dimensional (3D) model that incorporates an endothelial lumen, a 3D extracellular matrix, and an intact bacterial source to model the host microenvironment. Using this system, we show that an endothelial lumen significantly increased neutrophil migration toward a source of Pseudomonas aeruginosa Surprisingly, we found neutrophils, which were thought to be short-lived cells in vitro, migrate for up to 24 hours in 3D in the presence of an endothelial lumen and bacteria. In addition, we found that endothelial cells secrete inflammatory mediators induced by the presence of P aeruginosa, including granulocyte-macrophage colony-stimulating factor (GM-CSF), a known promoter of neutrophil survival, and interleukin (IL)-6, a proinflammatory cytokine. We found that pretreatment of neutrophils with a blocking antibody against the IL-6 receptor significantly reduced neutrophil migration to P aeruginosa but did not alter neutrophil lifetime, indicating that secreted IL-6 is an important signal between endothelial cells and neutrophils that mediates migration. Taken together, these findings demonstrate an important role for endothelial paracrine signaling in neutrophil migration and survival.

An Accessible Organotypic Microvessel Model Using iPSC-Derived Endothelium.

While organotypic approaches promise increased relevance through the inclusion of increased complexity (e.g., 3D extracellular microenvironment, structure/function relationships, presence of multiple cell types), cell source is often overlooked. Induced pluripotent stem cell (iPSC)-derived cells are potentially more physiologically relevant than cell lines, while also being less variable than primary cells, and recent advances have made them commercially available at costs similar to cell lines. Here, the use of induced pluripotent stem cell-derived endothelium for the generation of a functional microvessel model is demonstrated. High precision structural and microenvironmental control afforded by the design approach synergizes with the advantages of iPSC to produce microvessels for modeling endothelial biology in vitro. iPSC microvessels show endothelial characteristics, exhibit barrier function, secrete angiogenic and inflammatory mediators, and respond to changes in the extracellular microenvironment by altering vessel phenotype. Importantly, when deployed in the investigation of neutrophils during innate immune recruitment, the presence of the iPSC endothelial vessel facilitates neutrophil extravasation and migration toward a chemotactic source. Relevant cell sources, such as iPSC, combine with organotypic models to open the way for improved and increasingly accessible in vitro tissue, disease, and patient-specific models.

Chemokine Signaling and the Regulation of Bidirectional Leukocyte Migration in Interstitial Tissues.

Motile cells navigate through complex tissue environments that include both attractive and repulsive cues. In response to tissue wounding, neutrophils, primary cells of the innate immune response, exhibit bidirectional migration that is orchestrated by chemokines and their receptors. Although progress has been made in identifying signals that mediate the recruitment phase, the mechanisms that regulate neutrophil reverse migration remain largely unknown. Here, we visualize bidirectional neutrophil migration to sterile wounds in zebrafish larvae and identify specific roles for the chemokine receptors Cxcr1 and Cxcr2 in neutrophil recruitment to sterile injury and infection. Notably, we also identify Cxcl8a/Cxcr2 as a specific ligand-receptor pair that orchestrates neutrophil chemokinesis in interstitial tissues during neutrophil reverse migration and resolution of inflammation. Taken together, our findings identify distinct receptors that mediate bidirectional leukocyte motility during interstitial migration depending on the context and type of tissue damage in vivo.

Leading from the Back: The Role of the Uropod in Neutrophil Polarization and Migration.

Cell motility is required for diverse biological processes including development, homing of immune cells, wound healing, and cancer cell invasion. Motile neutrophils exhibit a polarized morphology characterized by the formation of leading-edge pseudopods and a highly contractile cell rear known as the uropod. Although it is known that perturbing uropod formation impairs neutrophil migration, the role of the uropod in cell polarization and motility remains incompletely understood. Here we discuss cell intrinsic mechanisms that regulate neutrophil polarization and motility, with a focus on the uropod, and examine how relationships among regulatory mechanisms change when cells change their direction of migration.

Effect of M1-M2 Polarization on the Motility and Traction Stresses of Primary Human Macrophages.

Macrophages become polarized by cues in their environment and this polarization causes a functional change in their behavior. Two main subsets of polarized macrophages have been described. M1, or "classically activated" macrophages, are pro-inflammatory and M2, or "alternatively activated" macrophages, are anti-inflammatory. In this study, we investigated the motility and force generation of primary human macrophages polarized down the M1 and M2 pathways using chemokinesis assays and traction force microscopy on polyacrylamide gels. We found that M1 macrophages are significantly less motile and M2 macrophages are significantly more motile than unactivated M0 macrophages. We also showed that M1 macrophages generate significantly less force than M0 or M2 macrophages. We further found that M0 and M2, but not M1, macrophage force generation is dependent on ROCK signaling, as identified using the chemical inhibitor Y27632. Finally, using the chemical inhibitor blebbistatin, we found that myosin contraction is required for force generation by M0, M1, and M2 macrophages. This study represents the first investigation of the changes in the mechanical motility mechanisms used by macrophages after polarization.

Integrin associated proteins differentially regulate neutrophil polarity and directed migration in 2D and 3D.

Directed neutrophil migration in blood vessels and tissues is critical for proper immune function; however, the mechanisms that regulate three-dimensional neutrophil chemotaxis remain unclear. It has been shown that integrins are dispensable for interstitial three-dimensional (3D) leukocyte migration; however, the role of integrin regulatory proteins during directed neutrophil migration is not known. Using a novel microfluidic gradient generator amenable to 2D and 3D analysis, we found that the integrin regulatory proteins Kindlin-3, RIAM, and talin-1 differentially regulate neutrophil polarization and directed migration to gradients of chemoattractant in 2D versus 3D. Both talin-1-deficient and RIAM-deficient neutrophil-like cells had impaired adhesion, polarization, and migration on 2D surfaces whereas in 3D the cells polarized but had impaired 3D chemotactic velocity. Kindlin-3 deficient cells were able to polarize and migrate on 2D surfaces but had impaired directionality. In a 3D environment, Kindlin-3 deficient cells displayed efficient chemotaxis. These findings demonstrate that the role of integrin regulatory proteins in cell polarity and directed migration can be different in 2D and 3D.

Macrophage motility is driven by frontal-towing with a force magnitude dependent on substrate stiffness.

The ability of macrophages to properly migrate is crucial to their success as early responders during the innate immune response. Furthermore, improper regulation of macrophage migration is known to contribute to several pathologies. The signaling mechanisms underlying macrophage migration have been previously studied but to date the mechanical mechanism of macrophage migration has not been determined. In this study, we have created the first traction maps of motile primary human macrophages by observing their migration on compliant polyacrylamide gels. We find that the force generated by migrating macrophages is concentrated in the leading edge of the cell - so-called frontal towing - and that the magnitude of this force is dependent on the stiffness of the underlying matrix. With the aid of chemical inhibitors, we show that signaling through the RhoA kinase ROCK, myosin II, and PI3K is essential for proper macrophage force generation. Finally, we show that Rac activation by its GEF Vav1 is crucial for macrophage force generation while activation through its GEF Tiam1 is unnecessary.

Two-dimensional motility of a macrophage cell line on microcontact-printed fibronectin.

The ability of macrophages to migrate to sites of infection and inflammation is critical for their role in the innate immune response. Macrophage cell lines have made it possible to study the roles of individual proteins responsible for migration using molecular biology, but it has not been possible to reliably elicit the motility of macrophage cell lines in two dimensions. In the past, measurements of the motility of macrophage cell lines have been largely limited to transwell assays which provide limited quantitative information on motility and limited ability to visualize cell morphology. We used microcontact printing to create polydimethylsiloxane (PDMS) surfaces functionalized with fibronectin that otherwise support little macrophage adhesion. We used these surfaces to measure macrophage migration in two dimensions and found that these cells migrate efficiently in a uniform field of colony-stimulating factor-1, CSF-1. Knockdown of Cdc42 led to a nonstatistically significant reduction in motility, whereas chemical inhibition of PI3K activity led to a complete loss of motility. Inhibition of the RhoA kinase, ROCK, did not abolish the motility of these cells but caused a quantitative change in motility, reducing motility significantly on high concentrations of fibronectin but not on low concentrations. This study illustrates the importance of studying cell motility on well controlled materials to better understand the exact roles of specific proteins on cell migration. © 2014 Wiley Periodicals, Inc.