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Ibrutinib impairs anti-Aspergillus responses
tions (control, germinating conidia and LPS). and was not modified when comparing the M1 to M3 group. We next measured the concentration of IL-8 in whole blood col- lected from at M0, M1 and M3 and infected for 2 h by A. fumigatus germinating conidia. These results confirmed that ibrutinib therapy was related to a decrease of IL-8 production after Aspergillus challenge in the M1 group (Figure 3B).
Ibrutinib has no effect on neutrophil mobility and chemotaxis
Because migration to infected sites is a prerequisite to mounting an efficient anti-fungal response, we tested the ability of neutrophils to react to the chemoattractant fMLP (Figure 4A). Percentage of chemotaxis was defined as the number of neutrophils that reached the pore against the total number of neutrophils in the surround- ing area. As shown in Figure 4B, neutrophils derived from
A
treated and untreated patients displayed similar capacity to reach the reservoir containing fMLP, demonstrating that ibrutinib treatment did not alter the ability of neu- trophils to migrate and respond to the fMLP signal.
Engulfment and killing of Aspergillus fumigatus by neutrophils are impaired in patients receiving ibrutinib
Video-microscopy was used to investigate the behavior of neutrophils and fungi (at the germinating conidia stage) as well as their interactions. Preliminary experiments using Aspergillus exposed to voriconazole, an antifungal drug with fungicidal activity against Aspergillus, indicated that dying Aspergillus stained transiently with Sytox green dye. As opposed to human cells, the fluorescence of the dead fungi was lost after approximately 15-20 min (data not shown). We therefore determined Aspergillus killing as the Sytox staining of the fungus and its inability to grow
B
Figure 3. Production of interleukin 8 by neu- trophils is impaired during ibrutinib therapy. (A) Neutrophils from whole blood taken from patients before ibrutinib treatment, and 1 and 3 months after starting ibrutinib therapy were untreated (exposed to phosphate-buffered saline, PBS), or stimulated with Aspergillus ger- minating conidia or lipopolysaccharide for 4 h at 37°C. Brefeldin A was added after 30 min of stimulation. Cells were stained with membrane antibodies, permeabilized, stained with inter- leukin 8 (IL-8) antibody and then analyzed by flow cytometry. The results are expressed as the percentage of positive cells. (B) Whole blood samples were stimulated with Aspergillus germi- nating conidia for 2 h. The supernatants were collected and tested for IL8 concentration.
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