(311d) The Presence of an Endothelial Lumen Drives Fungal Germination and Neutrophil Migration in a Physiologically Relevant in Vitro Model of Infection

During an infection in vivo, neutrophils extravasate across the vascular endothelium and migrate through tissues to reach a site of infection. This early step in the innate immune response is critical for driving neutrophil recruitment and dysregulation of this process or absence of neutrophils can lead to chronic infections. The complex interactions of neutrophils, endothelial cells, and pathogens could be a target for therapeutic intervention but the effect of an endothelial lumen on microbial and neutrophil functions is not known. Although in vivo models intrinsically account for multicellular interactions, their inherent complexities make it difficult to investigate the individual roles of these interactions. Therefore, physiologically relevant in vitro models are needed to study the neutrophil response to infection.

We previously developed and published an in vitro model of bacterial infection consisting of a model endothelial blood vessel, an extracellular matrix, and a source of live bacteria. Using this model, we found that neutrophils migrating out of an endothelial lumen in the presence of the bacteria Pseudomonas aeruginosa migrate further and survive longer than neutrophils in a lumen without an endothelium. In the current study, we used this physiologically relevant model to investigate how multicellular interactions affect the neutrophil response to the common fungal pathogen, Aspergillus fumigatus.

Humans are regularly exposed to airborne spores of the fungus Aspergillus and yet healthy individuals rarely develop Aspergillus infections. In contrast, immunocompromised individuals, such as leukemia patients and organ-transplant recipients, are at risk of developing invasive aspergillosis, a serious fungal infection that is difficult to treat and is associated with high mortality rates. Therefore, a better understanding of the mechanisms that drive the innate immune response to Aspergillus is needed in order to develop better therapeutic options. Neutropenia, or a lack of neutrophils, is a key risk factor in the development of invasive aspergillosis. Furthermore, the Aspergillus species Aspergillus fumigatus is responsible for the vast majority of aspergillosis cases in patients. Therefore, in this study, we sought to investigate the mechanisms driving neutrophil recruitment to Aspergillus fumigatus.

In our microscale infection model, endothelial lumens were fabricated in a collagen matrix using the LumeNEXT system. Aspergillus fumigatus spores were added to the top of the device and incubated for 6 hours. Primary human neutrophils were then isolated and seeded inside the lumens. Neutrophil migration and hyphal growth were visualized using time-lapse microscopy and quantified.

We first characterized A. fumigatus growth and found that, in our device, spores began to germinate into hyphae 14 hours after seeding. The hyphae continued to extend towards the endothelial lumen, demonstrating invasive hyphal growth, and eventually punched through the vessel wall about 8 hours after germination. Interestingly, our data suggests that signaling between the endothelium and fungus is required for efficient fungal germination and hyphal growth. While spores seeded in devices with an endothelial lumen germinated and formed a hyphal network, spores seeded in devices without an endothelial lumen did not germinate during the time course of the experiment. Spores in these devices were seen to germinate 48-72 hours after seeding, indicating that the presence of an endothelium is not required for germination but rather increases the rate of germination.

We were next interested in investigating neutrophil recruitment to A. fumigatus hyphae in our device. We found that there was a low level of basal migration by neutrophils before hyphal growth began but most cells remained in the endothelial lumen. Following the growth of A. fumigatus hyphae, neutrophils quickly migrated out of the lumens and coated individual hyphae, often creating neutrophils swarms on individual hyphal branches. Interestingly, neutrophils remained in the endothelial lumens for up to 16 hours before migrating out towards the fungus. This extended neutrophil lifetime in vitro indicates that signaling from the endothelium increased neutrophil survival. We have also found that neutrophils migrate to the fungus Aspergillus nidulans, indicating that this response is not specific to one fungal strain.

We previously found that IL-6 secretion from the endothelial lumen contributed to increased neutrophil migration to a source of the bacteria Pseudomonas aeruginosa. Therefore, we hypothesized that this same signaling mechanism might be activated in the presence of fungus. Interestingly, blocking antibodies against the IL-6 receptor did not reduce neutrophil migration to A. fumigatus, indicating that the endothelial vessel is differentially activated in bacterial and fungal infections.

Taken together these results show a direct role for multicellular interactions in driving both pathogen growth and the neutrophil response to infections. Additionally, this study highlights the importance of studying infection and neutrophil migration using physiologically relevant in vitro models that rely on cell-cell communication rather than the addition of exogenous cell activators or chemoattractant peptides.