Dataset Information

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

ABSTRACT: 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.

SUBMITTER: McMinn PH

PROVIDER: S-EPMC7045365 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

McMinn Patrick H PH Hind Laurel E LE Huttenlocher Anna A Beebe David J DJ

Lab on a chip 20191002 21

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 componen ...[more]

PMID: 31576879

Similar Datasets

Project description:Electric fields are generated in vivo in a variety of physiologic and pathologic settings, including wound healing and immune response to injuries to epithelial barriers (e.g. lung pneumocytes). Immune cells are known to migrate towards both chemical (chemotaxis), physical (mechanotaxis) and electric stimuli (electrotaxis). Electrotaxis is the guided migration of cells along electric fields, and has previously been reported in T-cells and cancer cells. However, there remains a need for engineering tools with high spatial and temporal resolution to quantify EF guided migration. Here we report the development of an electrotaxis-on-chip (ETOC) platform that enables the quantification of dHL-60 cell, a model neutrophil-like cell line, migration toward both electrical and chemoattractant gradients. Neutrophils are the most abundant white blood cells and set the stage for the magnitude of the immune response. Therefore, developing engineering tools to direct neutrophil migration patterns has applications in both infectious disease and inflammatory disorders. The ETOC developed in this study has embedded electrodes and four migration zones connected to a central cell-loading chamber with migration channels [10 µm X 10 µm]. This device enables both parallel and competing chemoattractant and electric fields. We use our novel ETOC platform to investigate dHL-60 cell migration in three biologically relevant conditions: 1) in a DC electric field; 2) parallel chemical gradient and electric fields; and 3) perpendicular chemical gradient and electric field. In this study we used differentiated leukemia cancer cells (dHL60 cells), an accepted model for human peripheral blood neutrophils. We first quantified effects of electric field intensities (0.4V/cm-1V/cm) on dHL-60 cell electrotaxis. Our results show optimal migration at 0.6 V/cm. In the second scenario, we tested whether it was possible to increase dHL-60 cell migration to a bacterial signal [N-formylated peptides (fMLP)] by adding a parallel electric field. Our results show that there was significant increase (6-fold increase) in dHL60 migration toward fMLP and cathode of DC electric field (0.6V/cm, n=4, p-value<0.005) vs. fMLP alone. Finally, we evaluated whether we could decrease or re-direct dHL-60 cell migration away from an inflammatory signal [leukotriene B4 (LTB4)]. The perpendicular electric field significantly decreased migration (2.9-fold decrease) of dHL60s toward LTB4 vs. LTB4 alone. Our microfluidic device enabled us to quantify single-cell electrotaxis velocity (7.9 µm/min ± 3.6). The magnitude and direction of the electric field can be more precisely and quickly changed than most other guidance cues such as chemical cues in clinical investigation. A better understanding of EF guided cell migration will enable the development of new EF-based treatments to precisely direct immune cell migration for wound care, infection, and other inflammatory disorders.

Project description:IntroductionAs 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.MethodsTo 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.ResultsCollagen 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.DiscussionOur 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.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets