Unleashing a Swarm: Characterizing the Mechanisms by which Neutrophil Swarming Restricts Fungal Hyphae

Additional Authors

Kelsey Owens, Department of Natural Sciences, University of Virginia at Wise, Wise, VA. Tammy R. Ozment, Department of Surgery, Quillen College of Medicine, East Tennessee State University, Johnson City, TN. Allison Scherer, Department of Natural Sciences, University of Virginia at Wise, Wise, VA.

Abstract

Neutrophils, a critical effector cell of innate immunity, utilize many mechanisms to combat infection. These cells employ a strong arsenal of tools, such as phagocytosis, NET production, and reactive oxygen species production, to eliminate pathogens within the body. Neutrophil swarming, a collective process of many neutrophils synchronizing their activities to target a single pathogen, has also been recently characterized. Although the molecular events triggering swarming have been characterized against artificial clusters of Canida albicans yeast, the biological relevance to this function remains unclear. We hypothesized that C. albicans hyphae, a larger morphology that is highly relevant to human disease and virulence during infection, would be a potent swarm inducing target and therefore a better system to elucidate the molecular mechanisms engaged during swarming. Unfortunately, swarming events against fungal hyphae are poorly characterized. To address this knowledge gap, we leveraged a microscale printing assay to target fungal hyphae and elucidate molecular pathways that neutrophils might use to induce swarming. To do this, 8x8 grids of Poly-l-lysine spots were printed using a Picospotter (PolyPico, Galway, Ireland) onto glass slides and C. albicans hyphae were attached to each spot. Confocal timelapse microscopy was used to monitor human neutrophil responses against these targets. Isolated human neutrophils were treated with inhibitors to target specific signaling pathways that are important for swarming function. We found that hyphae were indeed able to induce potent swarming responses. We found that by inhibiting spleen tyrosine kinase (SYK), Bruton’s tyrosine kinase (BTK), or cellular receptor CD18, neutrophils were unable to either induce or maintain swarming responses, which compromised the restriction of hyphal growth. Lastly, we observed interesting insights into pioneer neutrophil interactions with hyphal cells. This assay allowed us to further understand the biology behind neutrophil swarming and contribute to understanding the role swarming plays against infectious fungal pathogens

Start Time

16-4-2025 1:30 PM

End Time

16-4-2025 4:00 PM

Presentation Type

Poster

Presentation Category

Science, Technology and Engineering

Student Type

Graduate Student - Doctoral

Faculty Mentor

Alex Hopke

Faculty Department

Biomedical Sciences

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Apr 16th, 1:30 PM Apr 16th, 4:00 PM

Unleashing a Swarm: Characterizing the Mechanisms by which Neutrophil Swarming Restricts Fungal Hyphae

Neutrophils, a critical effector cell of innate immunity, utilize many mechanisms to combat infection. These cells employ a strong arsenal of tools, such as phagocytosis, NET production, and reactive oxygen species production, to eliminate pathogens within the body. Neutrophil swarming, a collective process of many neutrophils synchronizing their activities to target a single pathogen, has also been recently characterized. Although the molecular events triggering swarming have been characterized against artificial clusters of Canida albicans yeast, the biological relevance to this function remains unclear. We hypothesized that C. albicans hyphae, a larger morphology that is highly relevant to human disease and virulence during infection, would be a potent swarm inducing target and therefore a better system to elucidate the molecular mechanisms engaged during swarming. Unfortunately, swarming events against fungal hyphae are poorly characterized. To address this knowledge gap, we leveraged a microscale printing assay to target fungal hyphae and elucidate molecular pathways that neutrophils might use to induce swarming. To do this, 8x8 grids of Poly-l-lysine spots were printed using a Picospotter (PolyPico, Galway, Ireland) onto glass slides and C. albicans hyphae were attached to each spot. Confocal timelapse microscopy was used to monitor human neutrophil responses against these targets. Isolated human neutrophils were treated with inhibitors to target specific signaling pathways that are important for swarming function. We found that hyphae were indeed able to induce potent swarming responses. We found that by inhibiting spleen tyrosine kinase (SYK), Bruton’s tyrosine kinase (BTK), or cellular receptor CD18, neutrophils were unable to either induce or maintain swarming responses, which compromised the restriction of hyphal growth. Lastly, we observed interesting insights into pioneer neutrophil interactions with hyphal cells. This assay allowed us to further understand the biology behind neutrophil swarming and contribute to understanding the role swarming plays against infectious fungal pathogens