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The human pathogen Aspergillus (A.) fumigatus is a fungal mold that can cause severe infections in immunocompromised hosts. Pathogen recognition and immune cell cross-talk are essential for clearing fungal infections efficiently. Immune cell interactions in particular may enhance individual cell activation and cytotoxicity towards invading pathogens. This study analyzed the reciprocal cell activation of natural killer (NK) cells and monocyte-derived dendritic cells (moDCs) after stimulation with A. fumigatus cell wall fractions and whole-cell lysates. Furthermore, the impact of the on moDCs expressed fungal receptors Dectin-1 and TLR-2 on NK cell activation was analyzed. Stimulation of moDCs with ligands for Dectin-1 and TLR-2 and transfer of soluble factors on autologous NK cells showed that moDCs could induce NK cell activation solely by secreting factors. In summary, both cell types could induce reciprocal cell activation if the stimulated cell type recognized fungal morphologies and ligands. However, moDCs displayed a broader set of A. fumigatus receptors and, therefore, could induce NK cell activation when those were not activated by the stimulus directly. Consequently, new fungal receptors should be identified on NK cells. The NK cell characterization marker CD56 was reduced detected in flow cytometry after fungal co-culture. Notably, this decreased detection was not associated with NK cell apoptosis, protein degradation, internalization, or secretion of CD56 molecules. CD56 was shown to tightly attach to hyphal structures, followed by its concentration at the NK-A. fumigatus interaction site. Actin polymerization was necessary for CD56 relocalization, as pre-treatment of NK cells with actin-inhibitory reagents abolished CD56 binding to the fungus. Blocking of CD56 suppressed fungal mediated NK cell activation and secretion of the immune-recruiting chemokines MIP-1α, MIP-1β, and RANTES, concluding that CD56 is functionally involved in fungal recognition by NK cells. CD56 binding to fungal hyphae was inhibited in NK cells obtained from patients during immune-suppressing therapy after allogeneic stem cell transplantation (alloSCT). Additionally, reduced binding of CD56 correlated with decreased actin polymerization of reconstituting NK cells challenged with the fungus. The immune-suppressing therapy with corticosteroids negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES in NK cells after fungal stimulation ex vivo. Similar results were obtained when NK cells from healthy donors were treated with corticosteroids prior to fungal co-culture. Thus, corticosteroids were identified to have detrimental effects on NK cell function during infection with A. fumigatus., Der humanpathogene Pilz Aspergillus (A.) fumigatus kann lebensbedrohliche Infek-tionen in immunsupprimierten Patienten verursachen. Die Immunerkennung von Patho-genen sowie die Wechselwirkungen zwischen Immunzellen sind essentiell für die erfolg-reiche Bekämpfung von Pilzinfektionen. Zell-Zell-Interaktionen tragen zur gegenseitigen Aktivierung bei und können somit die Zytotoxizität gegenüber eindringenden Pathogenen steigern. In dieser Arbeit wurde die gegenseitige Zellaktivierung von natürlichen Killerzellen (NK-Zellen) und die aus Monozyten generierten, dendritischen Zellen (DZ) nach Stimula-tion mit Zellwandfraktionen und Zelllysaten des Pilzes A. fumigatus analysiert. Des Weite-ren wurde der Einfluss dendritischer Rezeptoren auf die NK-Zellaktivierung untersucht. Die Stimulation mit Liganden für Dectin-1 und TLR-2 induzierte die Freisetzung löslicher Faktoren, welche ausreichend waren, um autologe NK-Zellen zu stimulieren. Zusammen-fassend ist zu sagen, dass DZ mehr Rezeptoren zur Pilzerkennung exprimieren und so-mit NK-Zellen auch dann aktivieren konnten, wenn diese, aufgrund fehlender Rezepto-ren, nicht stimuliert wurden. Basierend auf diesen Ergebnissen sollten neue Pathogen-Erkennungsrezeptoren auf NK-Zellen identifiziert werden. Der Charakterisierungsmarker CD56 zeigte nach fun-galer Kokultur eine reduzierte Detektion in durchflusszytometrischen Analysen. Die ver-minderte Proteindetektion von CD56 war nicht assoziiert mit Apoptose, Internalisation, Sekretion oder Proteindegradierung. Weitere Analysen bestätigten eine starke CD56-Bindung zu Pilzhyphen, gefolgt von einer Konzentration des Proteins an der NK-A. fumi-gatus Interaktionsstelle. Diese Relokalisation zeigte eine Abhängigkeit zu Aktin, da Zytos-kelett-Inhibitoren die CD56-Bindung am Pilz verhinderten. Eine spezifische Blockade von CD56 Rezeptoren reduzierte die Freisetzung der immun-rekrutierenden Chemokine MIP-1α, MIP-1β und RANTES, was folgern ließ, dass CD56 eine funktionelle Rolle in der Pil-zerkennung der NK-Zellen hat. NK-Zellen, die während einer Immunsuppressionstherapie aus Empfängern einer al-logenen Stammzelltransplantation (alloSZT) gewonnen wurden, zeigten eine inhibierte Bindung von CD56 an Pilzhyphen. Diese korrelierte mit einer reduzierten Aktinpolymeri-sation nach fungaler Stimulation. Weiterhin hemmte eine Immunsuppressionstherapie mit Corticosteroiden die Sekretion von MIP-1α, MIP-1β und RANTES in NK-Zellen, die mit Pilz ex vivo stimuliert wurden. Ähnliches war zu beobachten, wenn NK-Zellen von ge-sunden Spendern vor Pilzstimulation mit Corticosteroiden vorbehandelt wurden. Daraus folgend wurde den Corticosteroiden ein negativer Einfluss auf die NK-Zellfunktion bei Pilzinfektionen zugesprochen.

Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56\(^{bright}\)CD16\(^{dim}\) cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.

Dendritic cells (DCs) are antigen presenting cells which serve as a passage between the innate and the acquired immunity. Aspergillosis is a major lethal condition in immunocompromised patients caused by the adaptable saprophytic fungus Aspergillus fumigatus. The healthy human immune system is capable to ward off A. fumigatus infections however immune-deficient patients are highly vulnerable to invasive aspergillosis. A. fumigatus can persist during infection due to its ability to survive the immune response of human DCs. Therefore, the study of the metabolism specific to the context of infection may allow us to gain insight into the adaptation strategies of both the pathogen and the immune cells. We established a metabolic model of A. fumigatus central metabolism during infection of DCs and calculated the metabolic pathway (elementary modes; EMs). Transcriptome data were used to identify pathways activated when A. fumigatus is challenged with DCs. In particular, amino acid metabolic pathways, alternative carbon metabolic pathways and stress regulating enzymes were found to be active. Metabolic flux modeling identified further active enzymes such as alcohol dehydrogenase, inositol oxygenase and GTP cyclohydrolase participating in different stress responses in A. fumigatus. These were further validated by qRT-PCR from RNA extracted under these different conditions. For DCs, we outlined the activation of metabolic pathways in response to the confrontation with A. fumigatus. We found the fatty acid metabolism plays a crucial role, along with other metabolic changes. The gene expression data and their analysis illuminate additional regulatory pathways activated in the DCs apart from interleukin regulation. In particular, Toll-like receptor signaling, NOD-like receptor signaling and RIG-I-like receptor signaling were active pathways. Moreover, we identified subnetworks and several novel key regulators such as UBC, EGFR, and CUL3 of DCs to be activated in response to A. fumigatus. In conclusion, we analyze the metabolic and regulatory responses of A. fumigatus and DCs when confronted with each other.