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D. Blez et al.
CD16, CD62L, Dectin 1, TLR2 and TLR4 (BD Biosciences) and CD66b (Biolegend).
Measurement of neutrophil oxidative burst
Neutrophils contained in 500 μL heparinized whole-blood sam- ples were incubated with 123-dihydro-rhodamine (Life Technologies) for 5 min at 37°C, then stimulated with the afore- mentioned stimuli for 2 h at 37°C.
Intracellular cytokine analysis
Whole-blood samples (500 μL) were stimulated with the above-mentioned stimuli for 4 h at 37°C. Brefeldin A (Sigma- Aldrich) was added after 30 min of stimulation. Cells were stained with membrane antibodies, permeabilized using the Intracellular Fixation & Permeabilisation Buffer Set (eBioscience) and stained with the following anti-human antibodies: interleukin (IL)-8, IL-6, tumor necrosis factor alpha (TNFα) (BD Biosciences) and IL-1β (R&D Systems). The concentration of IL-8 was determined using a Duo-Set enyme-linked immunosorbent assay kit from R&D Systems (Minneapolis, MN, USA).
Video-microscopy experiments
Three thousand Aspergillus fumigatus conidia were seeded in black, 96-well clear-bottom plates (Greiner) and allowed to germi- nate. After two washes, 48,000 isolated neutrophils in RPMI medium and Sytox green (final concentration 2 μM) were added. Interactions were visualized during 16 h of co-culture at 37°C using a Zeiss Axio Z1 fluorescent microscope (Carl Zeiss, Germany). Images were processed and analyzed using Imaris® software. The chemotaxis assay was performed using IncuCyte® ClearView 96-Well Chemotaxis Plates. Purified neutrophils stained by Hoechst (final concentration 10 μM) were placed in the membrane insert and formyl-methionine-leucyl-phenylalanine (fMLP), as a chemo-attractant (final concentration 10 μM), was put in the reservoir plate. More details are given in the Online Supplement.
Statistical analysis
GraphPad Prism 6 was used for statistical analyses (GraphPad software, La Jolla, CA, USA).
Ethics
This non-interventional study was performed in accordance with national regulations regarding the use of human material from healthy volunteers and patients. The local medical ethics committee (CPP Ile de France IV) approved the study, which was conducted in accordance with the principles of the Declaration of Helsinki. Signed informed consent to participation in the study was obtained from all patients.
Results
Effect of ibrutinib on CD11b, CD62L and Dectin-1 expression on neutrophils
Neutrophils were gated according to size and granular- ity by forward versus side scatter (FSC vs. SSC) and iden- tified as CD66b+, CD15+, CD16+/low, CD14- cells (Figure 1A). Surface expression of CD11b and CD62L (L- selectin) was evaluated at baseline then following stimu- lation by germinating conidia or LPS. In association with CD18 (β2 integrin), CD11b forms the heterodimeric inte- grin complement receptor 3 (CR3), also called macrophage-1 antigen, which is not only involved in the adhesion and migration of leukocytes but has also been
recognized as a major receptor for β-glucan.12 CD11b is overexpressed at the surface of neutrophils after degran- ulation as it is contained in secondary and tertiary neu- trophil granules.13 CD62L is involved in transient tether- ing of the neutrophils to the endothelial surface. Shedding of CD62L marks activation of neutrophils. As previously reported,14 germinating conidia and LPS induced significant activation of neutrophils, as evi- denced by an increase in CD11b and a decrease in CD62L expression (Figure 1B and data not shown). Expressed as mean fluorescence intensity (MFI), mean CD11b surface expression of the M0 group increased 2- fold after stimulation with germinating conidia (9,053 vs. 4,414) and by 3-fold after LPS stimulation (13,392 vs. 4,414). CD11b surface expression on unstimulated neu- trophils decreased after starting ibrutinib treatment (n=17 patients) in comparison with the expression prior to treatment initiation (n=17 patients) (Figure 1B). However, no statistically significant difference was observed when a paired analysis of eight patients sam- pled at M0, M1 and M3 was done (Figure 1C). After stimulating whole blood with germinating conidia, the increase of CD11b surface expression in M1 patients was statistically not different from that of the M0 group. Expressed as MFI, mean CD11b surface expression of the M1 group increased 3.3-fold after germinating conidia stimulation (6,857 vs. 2,070) and by 6-fold after LPS stim- ulation (12,394 vs. 2,070). Basal CD62L expression tend- ed to increase over time with a statistically significant difference between M0 and M3 (Figure 1D) but CD62L shedding following stimulation by germinating conidia did not change over time (Figure 1E). Paired analysis of six patients sampled at M0, M1 and then M3 indicated no difference over time (Figure 1F). We also analyzed basal expression of Dectin-1, TLR2 and TLR4, which play important roles in antifungal defenses.15,16 Dectin-1 expression decreased over time but the difference was not statistically significant. No difference was observed for TLR2 and TLR4 (Figure 1G and data not shown). Collectively, these results suggest that neutrophils from patients receiving ibrutinib slightly altered CD11b sur- face expression but no other important immune recep- tors. In addition, ibrutinib-exposed neutrophils seemed to maintain their ability to trigger a marked response to Aspergillus and LPS stimuli.
Reactive oxygen species production after Aspergillus challenge is decreased in patients receiving ibrutinib
As previously reported,14 ROS production increased after 2 h of stimulation by germinating conidia or LPS plus fMLP (Figure 2A). ROS production by neutrophils sampled at M1 was statistically lower for all conditions, i.e. PBS control, Aspergillus stimulation and LPS stimula- tion (Figure 2A, B). Expressed as MFI, mean ROS produc- tion decreased at M1 by 51.5% for the basal PBS condi- tion (2,333 vs. 1,131; P<0.05), 51% after stimulation with germinating conidia (9,330 vs. 4,569; P<0.05) and 31.6% after LPS stimulation (4,008 vs. 2,742; P<0.05) in compar- ison with M0. The defect in ROS production persisted in neutrophils at M3. Paired analysis of samples collected at M0 and M1 after stimulation with germinating conidia or LPS was achievable in 12 and 9 patients, respectively. As shown in Figure 2C, ROS production decreased markedly after 1 month of ibrutinib treatment both before and after stimulation by Aspergillus conidia or LPS.
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haematologica | 2020; 105(2)