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D. Blez et al.
further. Because killing germinating conidia requires close contact, we focused on germinating conidia engulfed by neutrophils and calculated the percentage of killing as the number of dying Aspergillus as a proportion of the whole number of germinating conidia engulfed by neutrophils.
Aspergillus killing proceeds in two steps. First, neu- trophils engulf i.e. deform and bind firmly to Aspergillus, next they prevent its growth and ultimately they kill the fungus 60 to 120 min later (Figure 5A). First, we found that the proportion of neutrophils that could engulf Aspergillus decreased after 1 and 3 months of treatment with ibrutinib (Figure 5B). This defect was restored when Aspergillus conidia were opsonized by addition of human autologous serum (Figure 5B). Strikingly, exposure to
A
ibrutinib was associated with a major impairment of the ability of neutrophils to kill Aspergillus. The percentage of germinating conidia killed by neutrophils fell from 50% atM0to1%atM1and0.8%atM3(Figure5C,D). Opsonization by autologous serum had no effect on the killing defect (Figure 5D).
Neutrophils exposed to ibrutinib in vitro have altered responses to Aspergillus
Similar experiments were conducted on neutrophils obtained from healthy donors. Addition of ibrutinib was associated with decreased cell surface expression of CD11b both at baseline (8,143 vs. 4,396; 46% decrease; P<0.05) and after challenge with germinating conidia (13,298 vs. 9,811;
B
Figure 4. Ibrutinib therapy does not alter neu- trophil chemotaxis. Neutrophil migration assays were performed using IncuCyte® ClearView 96- Well Chemotaxis Plates (BioScience). Ten thou- sand purified neutrophils were aliquoted into the membrane insert, while formyl-methionine- leucyl-phenylalanine (fMLP) was added to the reservoir plate. Migration of neutrophils towards the reservoir from the insert was observed in real time over the course of 60 min. The migra- tion index was defined as the proportion of neu- trophils migrating into the pore compared with all neutrophils crossing a square zone of 260 x 260 μm centered by the pore of interest. (A) At the beginning of the experiment (T0), the pres- ence of fMLP (i) led to migration of neutrophils towards the pore after 60 minutes (T60) (ii), whereas the vehicle control has no effect (iii. iv). (B) Neutrophils collected from patients before (M0) or after 1 month (M1) and 3 months (M3) of ibrutinib therapy were submitted to the chemotaxis assay. Statistical analysis was per- formed by applying the Kruskall Wallis test with the Dunn multiple comparison post-test. ns: non-significant.
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haematologica | 2020; 105(2)