Figure 2.
Figure 2. Endosomally driven Btk responses during human macrophage infection with A fumigatus are required for optimal fungal growth control. (A) TLR9 engagement and BTK phosphorylation are required for NFAT activation in response to A fumigatus in hMDMS. hMDMs were pretreated with Ibrutinib (1 µM), the TLR9-blocking nucleotide ODN2088 (10 µM), or ODN nucleotide control (10 µM) for 1 hour. Macrophages were then infected with A fumigatus swollen conidia (MOI = 1) for 1 hour. Whole cell lysates were separated by SDS-PAGE, followed by western blotting. Membranes were probed with anti-NFATc1 and Histone H3 antibodies. (B-C) BTK mediates an endosomal NF-ĸB activation pathway in human macrophages. (B) Monocyte-derived macrophages were pretreated with Scramble or BTK-targeting siRNA (100 nM) for 72 hours and additionally with cytochalasin D (10 µM) or vehicle. Macrophages were stimulated with eGFP A fumigatus swollen conidia (MOI = 1) for 1 hour, and NFATc1 and NFKB 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. (C) 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: ns, not significant; *P < .05; NS, nonstimulated. (D) BTK is recruited to the membranes of endosomes containing swollen but not resting conidia in hMDMs. Representative confocal microscopy of monocyte-derived macrophages infected with eGFP expressing A fumigatus (green) (MOI = 1) at 45 minutes propidium iodide stained for BTK (red) and nuclei (blue) (original magnification ×60). (a) Nuclei; (b) BTK; (c) eGFP; (d) composite images. (E) Ibrutinib does not block A fumigatus phagocytosis. Monocyte-derived macrophages were pretreated with Ibrutinib (1 µM) for 1 hour. Macrophages were infected with biotinylated A fumigatus-GFP (green fluorescent protein) swollen conidia (MOI = 1) for 2 hours. External conidia were then counterstained with Cy3 biotin antibody, and phagocytosis was measured by flow cytometry. (F) Ibrutinib impairs macrophage control of fungal growth in vitro. Monocyte-derived macrophages were pretreated with Ibrutinib (1 µM) for 1 hour. Cells were stimulated with A fumigatus swollen conidia (MOI = 1) for 6 hours. Galactomannan levels were measured in the tissue culture supernatants. UT, untreated.

Endosomally driven Btk responses during human macrophage infection with A fumigatus are required for optimal fungal growth control. (A) TLR9 engagement and BTK phosphorylation are required for NFAT activation in response to A fumigatus in hMDMS. hMDMs were pretreated with Ibrutinib (1 µM), the TLR9-blocking nucleotide ODN2088 (10 µM), or ODN nucleotide control (10 µM) for 1 hour. Macrophages were then infected with A fumigatus swollen conidia (MOI = 1) for 1 hour. Whole cell lysates were separated by SDS-PAGE, followed by western blotting. Membranes were probed with anti-NFATc1 and Histone H3 antibodies. (B-C) BTK mediates an endosomal NF-ĸB activation pathway in human macrophages. (B) Monocyte-derived macrophages were pretreated with Scramble or BTK-targeting siRNA (100 nM) for 72 hours and additionally with cytochalasin D (10 µM) or vehicle. Macrophages were stimulated with eGFP A fumigatus swollen conidia (MOI = 1) for 1 hour, and NFATc1 and NFKB 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. (C) 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: ns, not significant; *P < .05; NS, nonstimulated. (D) BTK is recruited to the membranes of endosomes containing swollen but not resting conidia in hMDMs. Representative confocal microscopy of monocyte-derived macrophages infected with eGFP expressing A fumigatus (green) (MOI = 1) at 45 minutes propidium iodide stained for BTK (red) and nuclei (blue) (original magnification ×60). (a) Nuclei; (b) BTK; (c) eGFP; (d) composite images. (E) Ibrutinib does not block A fumigatus phagocytosis. Monocyte-derived macrophages were pretreated with Ibrutinib (1 µM) for 1 hour. Macrophages were infected with biotinylated A fumigatus-GFP (green fluorescent protein) swollen conidia (MOI = 1) for 2 hours. External conidia were then counterstained with Cy3 biotin antibody, and phagocytosis was measured by flow cytometry. (F) Ibrutinib impairs macrophage control of fungal growth in vitro. Monocyte-derived macrophages were pretreated with Ibrutinib (1 µM) for 1 hour. Cells were stimulated with A fumigatus swollen conidia (MOI = 1) for 6 hours. Galactomannan levels were measured in the tissue culture supernatants. UT, untreated.

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