(K-434) The Distinct Neutrophil Response to Diverse Bacterial Species

Introduction:: Neutrophils serve an important physiological role as the first line of defense against infection making them an ideal therapeutic target for treating bacterial infections that are either difficult to treat or antibiotic resistant. Bacterial infections can wreak havoc on the human immune system and are a major health concern due to their prevalence and the increase in antibiotic resistant strains. Upon infection, neutrophils become activated by signals from both the pathogen and host cells, including the blood vessel endothelium, and then extravasate through the blood vessel and migrate to the site of infection. There, they perform antimicrobial functions including reactive oxygen species (ROS) production to clear the infection. It has been shown that the extent of the neutrophil response varies in the presence of distinct pathogen sources. While the neutrophil response to infection is well-defined, how neutrophils differentially respond to diverse pathogens remains unclear. The objective of this study was to use a physiologically relevant, Infection-on-a-Chip device, including live, intact bacteria, a 3D endothelial cell lumen structure, and extracellular matrix, to determine how neutrophils respond to diverse, clinically significant bacterial species, namely Pseudomonas aeruginosa, Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus. They are all commonly used as model infections for human immunology research and there is a pressing need to better equip our bodies to fight each of these infections. Specifically, S. aureus is frequently implicated worldwide for a role in morbidity and mortality as a cause of sepsis and pneumonia and is the most common multi-drug resistant bacterial species.

Materials and Methods:: Primary human neutrophils were isolated from blood donations from healthy donors following an approved IRB. Isolation was done using the MACSxpress neutrophil isolation kit (Miltenyi Biotech). The Infection-on-a-Chip devices were made following the previously characterized LumeNEXT system with Human Umbilical Vein Endothelial Cells (HUVECs) seeded onto a collagen surface to form a blood vessel lumen. Neutrophils were flowed into the lumen and bacteria added into the collagen to complete the infectious microenvironment. All imaging was conducted using an inverted microscope (Nikon eclipse Ti2) and a Nikon 10x/0.45 (NA) objective. Extravasation was measured by counting the number of cells outside the lumen over time and normalized to the initial neutrophil count in the device. Intracellular ROS production was quantified using DHR123 as a marker. The percentage of neutrophils expressing ROS was quantified in both the Infection-on-a-Chip device and in a well plate with collagen but no endothelial cells. Neutrophil migration outside of the lumen was tracked using MTrackJ software in Fiji. Speed, distance migrated (distance from start to end point), length of tracks, and straightness of migration (the distance migrated divided by the length of the track) were quantified. We used a multiplexed ELISA assay to determine which signals from the endothelium were contributing to neutrophil function in our device. All data were pooled from three or more independent replicates. Statistical analysis was conducted using R. The analysis accounts for variability within and between replicates and considers independent replicates individually. For each condition, estimated marginal means and standard error were calculated.

Results, Conclusions, and Discussions:: To study how neutrophils distinctly respond to diverse bacterial species, we started by investigating all steps of the neutrophil response: extravasation, migration, and antimicrobial ROS production. We found neutrophils extravasated from the lumen in response to each bacterial species; with significantly more neutrophils extravasating to L. monocytogenes. Additionally, S. aureus showed significantly lower extravasation. Using a multiplexed ELISA, we found endothelial secretion of IL-6 was upregulated in response to all bacterial species and using blocking antibodies determined it was universally important for neutrophil extravasation. We found similar levels of interstitial migration across bacteria. We were next interested in determining how neutrophil ROS production would differ between bacterial species in the Infection-on-a-Chip device. We found neutrophils produce intracellular ROS in response to each bacterial species in both a well plate and in the Infection-on-a-Chip device (Fig 1). We found a significantly higher number of neutrophils producing intracellular ROS in the presence of P. aeruginosa and S. enterica, gram-negative bacteria, compared to neutrophils in the presence of L. monocytogenes and S. aureus, gram-positive bacteria (Fig 1A). Intriguingly, we showed an increase in ROS production for each bacterial species following extravasation through an endothelium (Fig 1B) compared to without an endothelium (Fig 1C) demonstrating an endothelial role in neutrophil ROS production.

In this study, we demonstrated the ability of our Infection-on-a-Chip device to model neutrophil immune function while also elucidating differences in this response in the presence of diverse bacterial species. We showed the importance of the endothelium for continuing to research and understand neutrophil ROS production. Of note, an overall lack of neutrophil response towards S. aureus infection in our device mimics infections in humans where it is known to effectively evade neutrophil clearance. This device could be developed into an important experimental platform for studying neutrophil interactions with S. aureus and used to test treatments for MRSA patients. The impact of this study is an increased understanding of the neutrophil response to diverse bacterial species, leading to better targeted therapeutics and improved outcomes for patients infected with these globally harmful pathogens.


Acknowledgements (Optional): : This work was supported by funding from NIH 1R35GM146737-01.

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