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

Abstract

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.

Keywords: antimicrobial functions; cell-cell interactions; in vitro models; infection; inflammation; innate immunity; microfluidics; neutrophil.

Figure 1

Neutrophil Response to Infection. Following infection, endothelial cells lining the vasculature become activated, releasing adhesion molecules and cytokines. These signals activate neutrophils, initiating the leukocyte adhesion cascade. Neutrophils then extravasate through the blood vessel and migrate to the site of infection following PAMPs, released by the pathogen, and DAMPs released by tissue resident cells (macrophages, dendritic cells, fibroblasts). There, they fight the infection by releasing NETs and Reactive Oxygen Species (ROS), and directly phagocytosing the pathogen.

Figure 2

In Vitro Systems for Studying the Neutrophil Response to Infection. (A) Transwell assays, a well-in-well system with a porous membrane divider, are used to investigate neutrophil migration to chemokines (top, green gradient) and bacterial sources (bottom, red gradient) through cellular monolayers. (B) 2D microfluidic devices are used to investigate various aspects of neutrophil migration, including neutrophil reverse migration and migration through bifurcations, to soluble chemokines (top). Devices have also been designed to investigate direct interactions between neutrophils and both bacterial and fungal pathogens (bottom). (C) 3D microfluidic devices are used to investigate neutrophil migration to soluble chemokines in an extracellular matrix hydrogel following extravasation through an endothelium. Neutrophils migrate through an endothelial monolayer, seeded on the hydrogel, and into the ECM. (D) Organotypic microfluidic devices include a model vasculature containing endothelial cells in a relevant lumen geometry. These devices use both chemokines and live pathogens to induce migration.

Figure 3

Interactions with Immune Cells Influences the Neutrophil Response. Cell-cell signaling between neutrophils and other immune cells plays a significant role in the innate immune response to infection. Leading neutrophils influence swarming and the directional migration of trailing neutrophils (top middle). Monocytes induce neutrophil migration and in turn, neutrophils inhibit pro-inflammatory signaling by monocytes (top right). Release of miR-146a rich exosomes induces neutrophil extracellular trap formation and reactive oxygen species generation (bottom right). Dendritic cells stimulate neutrophil migration while neutrophils have a dual effect on dendritic cells, stimulating migration through release of α-defensins while reducing DC production of inflammatory signals through signaling through NETs (bottom middle). NK cells can both promote neutrophil survival following stimulation by pro-inflammatory cytokines and promote neutrophil apoptosis following stimulation with anti-inflammatory cytokines (bottom left). Neutrophils stimulate Th17 cell migration and induce CD4 T cells to become Th17 cells, which in turn stimulate neutrophil migration (top left).