(600c) Heterotypic Interactions with an Endothelial Lumen Increase Neutrophil Lifetime and Migration to Pseudomonas Aeruginosa Via IL6 Signaling

During an infection, neutrophils migrate across the vascular endothelium, leaving the blood vessel to reach sites of infection. This early step in the innate immune response is critical for pathogen clearance, with improper regulation of this process leading to chronic infections or inflammation. While many of the signaling pathways involved in endothelial capture and extravasation of neutrophils are known, the role of the endothelium in promoting neutrophil migration to a site of infection remains unclear. To address this question, we have developed an in vitro model of neutrophil recruitment that incorporates many aspects of an in vivo infection including a model endothelial blood vessel, the extracellular matrix, and a source of live bacteria and used this model to study endothelial cell-neutrophil interactions and neutrophil migration in an infectious environment.

In our microscale infection model, endothelial lumens were fabricated in a collagen matrix using the characterized LumeNEXT system. Primary human neutrophils were isolated and seeded in the lumens. Neutrophil migration was then initiated with a gradient of the bacteria Pseudomonas aeruginosa. Migration was visualized using time-lapse microscopy and the total number of migrated neutrophils and the migration distance were used to quantify migration over time.

We first characterized our infection model and determined that the P. aeruginosa diffused throughout the matrix after about one hour but stopped at the lumen edge. Furthermore, using FITC-dextran to measure diffusion, we found that endothelial vessels in the presence of P. aeruginosa were leakier than without P. aeruginosa but still had good barrier function. We also confirmed that neutrophils were only able to migrate out of the vessels to a source of P. aeruginosa, not culture media alone. To determine the role of the endothelium in neutrophil migration, we visualized neutrophils migrating out of lumens with and without an endothelial barrier. We found that neutrophils migrating out of an endothelial lumen to a source of P. aeruginosa have significantly increased migration and lifetime compared to neutrophils that do not migrate through an endothelium. Endothelial lined lumens had vastly more migrated neutrophils and the neutrophils migrate significantly farther compared to unlined lumens. This migration was dependent on known adhesion signals as pretreatment with a blocking antibody against B₂ integrin significantly reduced neutrophil migration. Interestingly, neutrophils in our system migrated for up to 24 hours after being seeded in an endothelial lumen, which is surprising as it is commonly accepted that neutrophils are short-lived cells, with half-lives of only a few hours. Using the live/dead stain propidium iodide we confirmed that neutrophils migrating through an endothelial lumen have increased survival compared to those without an endothelium. We were next interested in determining what factors endothelial cells produce in response to P. aeruginosa that promote neutrophil lifetime and migration. We used a custom multiplexed ELISA to compare secreted protein levels in media from the lumens in various conditions and we found that endothelial cells secrete several important inflammatory mediators in response to P. aeruginosa, including GM-CSF, a known promoter of neutrophils survival and IL-6. Pretreatment of neutrophils with a blocking antibody against the IL-6R significantly reduced their migration, indicating that secreted IL-6 is an important pro-migration signal between endothelial cells and neutrophils in an infection. Indicating that the endothelium produces IL-6 in the presence of P. aeruginosa and this is required for efficient neutrophil migration.