Environmental Dimensionality Controls the Interaction of Phagocytes with the Pathogenic Fungi Aspergillus fumigatus and Candida albicans

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Abstract
The fungal pathogens Aspergillus fumigatus and Candida albicans are major health threats for immune-compromised patients. Normally, macrophages and neutrophil granulocytes phagocytose inhaled Aspergillus conidia in the two-dimensional (2-D) environment of the alveolar lumen or Candida growing in tissue microabscesses, which are composed of a three-dimensional (3-D) extracellular matrix. However, neither the cellular dynamics, the per-cell efficiency, the outcome of this interaction, nor the environmental impact on this process are known. Live imaging shows that the interaction of phagocytes with Aspergillus or Candida in 2-D liquid cultures or 3-D collagen environments is a dynamic process that includes phagocytosis, dragging, or the mere touching of fungal elements. Neutrophils and alveolar macrophages efficiently phagocytosed or dragged Aspergillus conidia in 2-D, while in 3-D their function was severely impaired. The reverse was found for phagocytosis of Candida. The phagocytosis rate was very low in 2-D, while in 3-D most neutrophils internalized multiple yeasts. In competitive assays, neutrophils primarily incorporated Aspergillus conidia in 2-D and Candida yeasts in 3-D despite frequent touching of the other pathogen. Thus, phagocytes show activity best in the environment where a pathogen is naturally encountered. This could explain why “delocalized” Aspergillus infections such as hematogeneous spread are almost uncontrollable diseases, even in immunocompetent individuals. Aspergillus fumigatus and Candida albicans are the most common of all human pathogenic fungal germs. Normally, inhaled Aspergillus spores are destroyed by alveolar macrophages and polymorphonuclear neutrophils (PMNs), both of which are lung phagocytes, i.e., cells that kill inhaled microbes by ingestion. In contrast, C. albicans is a normal constituent of the human gut flora that is controlled by tissue-resident PMNs. If immune control is lost, both fungi grow into the surrounding tissue and cause life-threatening infections. To investigate how phagocytes function in the disparate environments of lung air sacs (lacking a definite matrix-composition [two-dimensional (2-D)]) or mucosal tissues (providing a three-dimensional [3-D] space), the authors mimicked 2-D and 3-D environments and analyzed the process of ingestion, called phagocytosis, by PMNs and other phagocytes. Phagocytosis was a dynamic cellular process where distinct cells showed vastly different behavior. The environmental setup (2-D versus 3-D) had a profound impact on the cell's ability to phagocytose. Aspergillus conidia were much better ingested in 2-D systems, while Candida yeasts were only ingested in 3-D systems, even if the other pathogen was present. This was true for different 2-D and 3-D systems and for both cells of mice and humans. Besides providing a comprehensive analysis of the cellular movements underlying phagocytosis, the results also suggest an evolution of phagocytes to optimally recognize fungal pathogens in the environment of natural infection.