Ibrutinib blocks Btk-dependent activation of NFAT and NF-κB in human macrophages during A fumigatus infection. (A) A fumigatus induces autophosphorylation of Btk at Tyr 223, and this is inhibited by Ibrutinib. THP1 macrophages were pretreated with Ibrutinib (1 µM) for 1 hour. Cells were stimulated with A fumigatus swollen conidia (multiplicity of infection [MOI] = 5) for up to 2 hours. Whole cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by western blotting. Membranes were probed with anti-pBTK and anti-BTK antibodies. (B) BTK phosphorylation is required for NFAT and NF-κB activation in response to A fumigatus in THP1 macrophages. THP1 macrophages were pretreated with Ibrutinib (1 µM) for 1 hour. Cells were stimulated with A fumigatus swollen conidia (MOI = 5) for up to 2 hours. Whole cell lysates were separated by SDS-PAGE, followed by western blotting. Membranes were probed with anti-NFATc1, NF-κB, and HDAC antibodies. (C-D) BTK mediates NFAT and NF-ĸB activation pathways in hMDMs (supplemental Figure 2). Monocyte-derived macrophages were pretreated with Scramble or BTK-targeting siRNA (75 nM) for 72 hours. Cells were stimulated with eGFP A fumigatus swollen conidia (MOI = 1) for 1 hour, and NFATc1 and NF-κB translocation were measured by confocal microscopy. Nuclear translocation was quantified by calculating the percent overlap of the nuclear DAPI and transcription factor-linked fluorophore channels. Data were calculated from 7 fields of view taken at random per biological repeat. Mean and standard deviation of 3 biological repeats are represented. Statistical analysis was performed using paired Student t tests: ns, not significant; *P < .05; NS, nonstimulated. N = 3. Af, A fumigatus; Ca, C albicans; Zym, zymosan. (E-H) TNF-α release by human macrophages in response to A fumigatus is BTK dependent. Monocyte-derived macrophages (E,G-H) and alveolar macrophages (F) were pretreated with Ibrutinib (1 µM) for 1 hour or scramble or BTK-targeting siRNA (100 nM) for 72 hours. (E-G) Macrophages were stimulated with A fumigatus swollen conidia (MOI = 1) for 16 hours. TNF-α levels in the tissue culture supernatants were measured by enzyme-linked immunosorbent assay. (H) Whole cell lysates were separated by SDS-PAGE, followed by western blotting. Membranes were probed with anti-BTK and anti–β-actin antibodies. Statistical analysis was performed using paired Student t tests. *P < .05. NS, nonstimulated. N = 3 to 5.