Person:

Ellett, Felix

Invasive aspergillosis (IA), primarily caused by Aspergillus fumigatus, is an opportunistic fungal infection predominantly affecting immunocompromised and neutropenic patients that is difficult to treat and results in high mortality. Investigations of neutrophil-hypha interaction in vitro and in animal models of IA are limited by lack of temporal and spatial control over interactions. This study presents a new approach for studying neutrophil-hypha interaction at single cell resolution over time, which revealed an evasive fungal behavior triggered by interaction with neutrophils: Interacting hyphae performed de novo tip formation to generate new hyphal branches, allowing the fungi to avoid the interaction point and continue invasive growth. Induction of this mechanism was independent of neutrophil NADPH oxidase activity and neutrophil extracellular trap (NET) formation, but could be phenocopied by iron chelation and mechanical or physiological stalling of hyphal tip extension. The consequence of branch induction upon interaction outcome depends on the number and activity of neutrophils available: In the presence of sufficient neutrophils branching makes hyphae more vulnerable to destruction, while in the presence of limited neutrophils the interaction increases the number of hyphal tips, potentially making the infection more aggressive. This has direct implications for infections in neutrophil-deficient patients and opens new avenues for treatments targeting fungal branching.

Neutrophils are key cellular components of the innate immune response and characteristically migrate from the blood towards and throughout tissues. Their migratory process is complex, guided by multiple chemoattractants released from injured tissues and microbes. How neutrophils integrate the various signals in the tissue microenvironment and mount effective responses is not fully understood. Here, we employed microfluidic mazes that replicate features of interstitial spaces and chemoattractant gradients within tissues to analyze the migration patterns of human neutrophils. We find that neutrophils respond to LTB4 and fMLF gradients with highly directional migration patterns and converge towards the source of chemoattractant. We named this directed migration pattern convergent. Moreover, neutrophils respond to gradients of C5a and IL-8 with a low-directionality migration pattern and disperse within mazes. We named this alternative migration pattern divergent. Inhibitors of MAP kinase and PI-3 kinase signaling pathways do not alter either convergent or divergent migration patterns, but reduce the number of responding neutrophils. Overlapping gradients of chemoattractants conserve the convergent and divergent migration patterns corresponding to each chemoattractant and have additive effects on the number of neutrophils migrating. These results suggest that convergent and divergent neutrophil migration-patterns are the result of simultaneous activation of multiple signaling pathways.