Interferon-γ (IFN-γ) Mediates a Direct Fungicidal Effect of Human Natural Killer (NK) Cells Against Aspergillus Fumigatus
Authors: Bouzani Maria , Ok Michael , Cormick Allison Mc , Ebel Frank , Einsele Hermann , Loeffler Juergen 
Published: October 2019

Abstract
Abstract 2779
NK cells are innate immune effector cells, which are well recognized for their role against cells transformed by malignant processes or by an intracellular infection. Although, there is increasing evidence of an interaction of NK cells also with extracellular pathogens, less is known about their interplay with moulds and especially with A. fumigatus. The interest for this interaction is mainly driven by the high mortality characterizing invasive aspergillosis (IA), an infection of immunosuppressed patients caused mainly by this fungus. In our study, we investigated the in vitro interaction of human NK cells with A. fumigatus. Methods. NK cells were magnetically depleted from the peripheral blood of healthy volunteers followed by pulsing for 24h with 500 U/ml recombinant interleukin–2 (rhIL-2). The induction of IFN-γ and Tumor Necrosis Factor-α (TNF-α) by the fungus was measured by real time RT-PCR and ELISA assays after 0, 3, 6 and 12h of co-culture. The lethal impact of NK cells and culture supernatants on A. fumigatus was evaluated by plate killing and 2,3-bis[2-methoxy-4-nitro-5-sulpho-phenyl]2H-tetrazolium-5-carboxanilide (XTT) assays. Transwell permeable membranes with pores of 0.4 μm, prohibiting the direct contact of cells placed on opposite sides but allowing the free circulation of molecules were used to evaluate the relevance of a direct cell – fungal contact on cytokine induction and fungal damage. The degranulation markers CD107a and CD107b were measured to assess the involvement of cytotoxic proteins on the anti-Aspergillus effect. The supernatant was magnetically depleted of IFN-γ. Recombinant human IFN-γ (rhIFN-γ) was used to mimic the effect of IFN-γ released by NK cells.

Results.
Co-cultivating human NK cells with resting (conidia) and germinated (germlings) fungal morphologies, only germlings induce a Th1 like cytokine response of NK cells, with the major representative IFN-γ (p<.05). Consistently, NK cells demonstrate a strong fungicidal effect only towards germlings (p<.05). Using transwell membranes to investigate the mechanism of this interaction, we found that physical contact was a prerequisite for the induction of cytokines (p<.05), but also for the release of a soluble factor, exhibiting fungicidal properties. Once the soluble factor was released, germlings were killed without the presence of NK cells (p<.05). To confirm this finding, germlings were incubated with supernatant obtained from the culture of NK cells with A. fumigatus. After 3 h of culture, 40% killing (p<.001) was demonstrated. To determine the soluble factor(s) causing the fungicidal effect, we explored the degranulation of cytotoxic proteins like perforin, granzymes and granulysin. FITC-labeled antibodies against the degranulation markers CD 107a, b were co-incubated with unstimulated NK cells, NK cells challenging germlings and NK cells challenging the cell line K562. In the presence of A. fumigatus, there was no expression of CD 107a, b, while the expression of these markers was significant in the culture with K562 cells. This result suggests that the granule proteins are not the means of the NK cell cytotoxicity against A. fumigatus. Moreover, the protective effect of IFN- γ for the host defense against IA is well known. So far, this effect was attributed to the influence of IFN- γ on innate immune cells, increasing their antifungal activity. In our study, we hypothesized that IFN- γ might demonstrate direct antifungal properties, mediating the fungicidal activity of NK cells. Thus, we compared the fungal killing caused by IFN- γ partially depleted supernatant and un-depleted supernatant and we found that the depletion of IFN- γ diminished the fungicidal effect (p<.05). Moreover, we exposed germlings to culture medium enriched with rhIFN- γ at concentrations equivalent to those detected in the supernatants and we observed a similar killing rates. To summarize, our study demonstrates that human NK cells interact with A. fumigatus germlings, which induce the release of Th1 like cytokines and cause significant fungal killing. Our data show for the first time that IFN-γ, released by NK cells, is a soluble molecule mediating direct fungicidal activity. These results attribute new characteristics to human NK cells, suggesting them as a potential alternative therapeutic tool against IA.

Disclosures:
No relevant conflicts of interest to declare.