(437f) Neutrophils Demonstrate Unique Functional Responses to Diverse Bacterial Pathogens in an Infection-on-a-Chip Microfluidic Model

As one of the first immune cells to respond to infection, neutrophils play a critical role in clearing pathogens and preventing disease. To accomplish this, neutrophils must efficiently respond to a variety of pathogens including different classes and strains of bacteria. Following infection, neutrophils become activated and undergo a generalized response in which they 1) migrate across the vascular endothelium through a process known as transendothelial migration, 2) migrate through the tissue to reach the site of infection, and 3) perform antimicrobial functions such as phagocytosis, NETosis, and reactive oxygen species generation to kill the pathogen. While the general steps in this process have been studied in great detail, the differential mechanisms used by neutrophils to respond to distinct bacterial pathogens remains unclear. One barrier to understanding has been a lack of physiologically relevant models that recapitulate the multiple steps of activation and function using human neutrophils. To overcome this challenge, we developed an infection-on-a-chip model that incorporates key aspects of the human infectious microenvironment including a model blood vessel, a 3D extracellular matrix, primary human neutrophils, and a source of live bacteria. In this study, we used our model to investigate how neutrophil extravasation, migration, and reactive oxygen species (ROS) generation varies in response to four bacterial species (Pseudomonas aeruginosa, Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus).These bacterial species were chosen as they span the range of gram-negative, gram-positive, intracellular, and extracellular pathogens and because they are all important pathogens in a clinical setting, with many of them falling on the World Health Organization’s list of priority pathogens.

To fabricate our infection-on-a-chip model, endothelial lumens were fabricated in collagen matrices to create model blood vessels and tissue structures. Primary human neutrophils were seeded in the lumens and each bacterial species was individually added to the bacterial port to create a gradient of bacteria perpendicular to the lumen edge. Neutrophil extravasation, migration, and ROS generation were visualized using time-lapse confocal microscopy.

We found significantly more neutrophils extravasated in response to L. monocytogenes compared to the other bacterial species. Interestingly, the rate of neutrophil extravasation was similar in response to L. monocytogenes and P. aeruginosa for the first 3 hours but while the normalized number of extravasated neutrophils plateaued in response to P. aeruginosa at 4 hours, the number of extravasated neutrophils continued to increase in response to L. monocytogenes for 10 hours. Interestingly, all responses required IL-6 signaling from the endothelium for an efficient response. We also investigated neutrophil migration in the tissue following extravasation in response to all four bacterial species. No strong patterns in neutrophil migration speed, length, and distance emerged between bacterial species but, intriguingly, neutrophils had significantly increased migration path straightness in response to S. aureus, potentially indicating a more directed response.

We also investigated neutrophil antimicrobial function in the form of ROS generation using DHR123 to monitor intracellular ROS. We found that neutrophil produced significantly more ROS in response to the two gram-negative bacterial species (P. aeruginosa and S. enterica). We initially discovered this for neutrophils seeded in collagen gels in a 48-well plate format but found the same trend in our infection-on-a-chip device. Interestingly, we found significantly more neutrophils expressed ROS in our infection-on-a-chip device than in the well format, indicating the presence of the endothelial lumen and activation via extravasation has a significant effect on neutrophil function.

Together, these results highlight our device’s potential to identify aspects of the neutrophil response (extravasation, migration, antimicrobial function) that are unique to a single pathogen or a class of bacteria and to determine which factors are universally important to the neutrophil response. Furthermore, it illustrates the importance of investigating the neutrophil response in a physiologically relevant model of the human infectious microenvironment.