(73f) Macrophage Regulation of Neutrophil Function in an Inflammation-on-a-Chip Microfluidic Model

Macrophages and neutrophils are both critical players in the innate immune response to infection. While these cells each perform unique roles in the clearance of pathogens, they also interact with each other through the secretion of soluble signals and the physical interactions. These interactions are not fully characterized, furthermore, the polarization of macrophages can alter how they influence the inflammatory microenvironment. Tools to analyze these interactions have been a significant hurdle as studying these cells in a relevant environment over relevant timescales is challenging in vitro and manipulating cellular phenotype or environment is difficult in vivo. In this study, we use our novel infection-on-a-chip model to investigate how macrophages modulate the neutrophil response to infection in a physiologically relevant environment.

To fabricate our infection-on-a-chip model, collagen I is polymerized around a sacrificial PDMS rod. This rod is then removed, leaving behind a hollow lumen vessel. This vessel is seeded with endothelial cells to create a model blood vessel. In our initial proof-of-concept study, we embedded THP-1 macrophages in the collagen, the tissue compartment, of our infection-on-a-chip device. These cells were differentiated in the collagen over 3 days prior to endothelial cell seeding. Primary human neutrophils were then seeded in the lumens and Pseudomonas aeruginosa was added to the bacterial port to create a source of infection. Neutrophil responses were visualized using time-lapse confocal microscopy.

Our initial study found that THP-1 macrophages survived and remained differentiated in the collagen. Furthermore, we showed that they took on a M2 phenotype in this experimental platform. We found no changes to the endothelial blood vessel structure or permeability in the presence of macrophages but showed significantly reduced neutrophil extravasation when macrophages were present, indicating a change to the inflammatory microenvironment.

We are now extending these studies to instead use primary human macrophages in our device. Furthermore, we are polarizing the macrophages into M1 and M2 phenotypes to understand the differential role of pro- and anti-inflammatory macrophages in modulating neutrophil function. To date, we have found that primary human macrophages are able to readily differentiate in collagen, in our device. They remain viable and polarize to M1/M2 phenotypes under the presence of LPS/IFN (M1) or IL-4 (M2). The presence of macrophages does not alter vessel structure or permeability. We are now running extravasation experiments to understand the role of M1 and M2 primary human macrophages in altering the neutrophil response. We are running these both under infectious and sterile inflammation conditions. Furthermore, we are investigating how the presence of macrophages alters the antimicrobial functions of neutrophils including the percentage of neutrophils undergoing NETosis as this process is a critical aspect of many inflammatory pathologies.