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D. Blez et al.
Neutrophils from ibrutinib-treated patients fail
to produce interleukin-8 upon Aspergillus stimulation
We evaluated intracellular production of IL-1β, IL-6, TNFα and IL-8 by neutrophils in whole blood by flow cytometry. In our experimental conditions, we did not detect changes in IL-1β, IL-6 or TNFα either before or after Aspergillus or LPS challenge over time (data not
AB
shown). We next focused on IL-8, a major chemoattractant for neutrophils. As shown in Figure 3A, the mean per- centage of IL-8-positive cells increased 4.7-fold (20.1% vs. 4.3%) and 9.7-fold (41.7% vs. 4.3%) after stimulation for 4 h with LPS or germinating conidia, respectively (Figure 3A). The percentage of IL-8-positive cells decreased in the M1 group in comparison with the M0 group for all condi-
C
Figure 2. Evolution of reactive oxygen species production by neutrophils during ibrutinib therapy. (A) Whole-blood neutrophils from a 61-year old man with chronic lymphoid leukemia were analyzed by flow cytometry for production of reactive oxygen species (ROS) in the basal condition (red) and after stimulation with 106 Aspergillus fumigatus germinating conidia (blue) before (upper panel) and after 62 days of ibrutinib therapy (lower panel). (B) ROS production by neu- trophils in whole blood collected from patients before ibrutinib therapy, 1 month after starting ibrutinib treatment and 3 months after initiation of ibrutinib ther- apy and stimulated with 106 Aspergillus germinating conidia or bacterial lipopolysaccharide plus formyl-methionine-leucyl-phenylalanine (LPS). Long bars represent the mean fluorescence intensity (MFI), and short bars represent the 10-90th percentile. The Kruskal-Wallis test with the Dunn multiple compar- ison post-test was applied *P<0.05; **P<0.01; ns: non-significant. (C) Paired analysis of ROS production by neutrophils in 500 μL whole blood from patients before and after 1 month of ibrutinib therapy under various conditions: (i) no stimulation (basal level); (ii) after stimulation with 106 Aspergillus fumigatus germinating conidia; or (iii) challenge with bacterial lipopolysaccharide plus formyl-methionine-leucyl-phenylalanine (LPS). Wilcoxon matched pairs test was applied; **P<0.01; ***P<0.005
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haematologica | 2020; 105(2)