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26.2% decrease; P<0.05) (Figure 6A). Interestingly, CD11b expression was unmodified after LPS stimulation (17,789 vs. 15,674; decrease: 11.9%; P=NS), suggesting that the effect of ibrutinib was specific to the antifungal response path- way. Ibrutinib also impaired ROS production in all condi- tions, as evidenced by decreases of 68.2% from basal level (1,205 vs. 383; P<0.005), 58.3% after challenge with germi- nating conidia (5,298 vs. 2,209; P<0.005) and 69% after LPS stimulation (4,834 vs. 1,500; P<0.005) (Figure 6B).
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Finally, we confirmed the video-microscopy observa- tions, using blood from healthy donors exposed to 5 μM ibrutinib for 10 min. Ibrutinib led to a decrease of engulf- ment ability and a dramatic impairment of Aspergillus killing by neutrophils (Figure 6C, D). Because we found that ibru- tinib markedly impaired ROS production, we examined whether the inhibition of ROS production by diphenyleneiodonium (DPI), a NADPH oxidase inhibitor, could lead to similar results. DPI caused not only a pre-
Figure 6. In vitro effect of ibrutinib on neutrophil reactive oxygen species production, CD11b sur- face expression and killing of Aspergillus fumigatus. Blood col- lected from six healthy donors was incubated for 10 min at 37°C with ibrutinib (final concentration of 1 μM or 5 μM) or dimethylsulfoxide (DMSO) vehicle as a control and then stimulated for 2 h before flow cytometry analysis. The long hori- zontal bars indicate the mean, and the short bars indicate the stan- dard error of the mean. (A) Expression of surface CD11b on neutrophils without stimulation (phosphate-buffered saline, PBS) or after stimulation with germinat- ing Aspergillus conidia or bacterial lipopolysaccharide (LPS) plus formyl-methionine-leucyl-pheny- lalanine (fMLP). (B) Reactive oxy- gen species (ROS) production by neutrophils without stimulation (PBS) or stimulated by germinating Aspergillus conidia or LPS plus fMLP. Neutrophils were purified from blood collected from five healthy donors and then incubat- ed for 10 min with ibrutinib (final concentration: 5 μM) or DMSO. (C) Engulfment of Aspergillus germi- nating conidia by neutrophils was observed in real-time by video- microscopy during co-culture for 16 h. (D) The rate of killing of Aspergillus germinating conidia by neutrophils was determined in real-time by video-microscopy dur- ing co-culture for 16 h. Blood col- lected from four healthy donors was incubated for 10 min with the NADPH oxidase inhibitor diphenyleneiodonium (DPI; final concentration 5 μM) or DMSO before stimulation or neutrophil purification. (E) ROS production by neutrophils in blood with or with- out DPI and stimulated for 2 h with germinating conidia, assessed by flow cytometry analysis. (F) Rate of killing of Aspergillus germinating conidia by neutrophils isolated from blood incubated with or with- out DPI determined in real-time by video-microscopy during a 16 h co- culture. Multiple group analysis was assessed by the Friedman test for paired data with the Dunn multiple comparison post-test; two-group analysis was assessed by a paired t-test; *P<0.05; **P<0.005; ***P<0.0005; ns: non-significant.
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haematologica | 2020; 105(2)