Fig. 2: Ibrutinib impairs M1 polarization and affects macrophage and γδ T cell response to Mycobacterium tuberculosis.

To obtain the M1 profile, monocytes from HD were cultured with GM-CSF (50 ng/ml) for 7 days and IFN-γ (10 ng/ml) was added for the last 2 days of culture. Ibrutinib (0.3 µM) was added to the culture 30 min before adding IFN-γ. a At day 7, macrophages were detached and stained with anti-CD14 PerCP/Cy5.5, anti-CD16 FITC, anti-CD86 PerCP/Cy5.5, anti-CD206 FITC, anti-CD163 PE, or anti-HLA-DR FITC and analyzed by using a FACScan flow cytometer (BD Immunocytometry Systems, San Jose, CA, USA). The results are shown as the mean fluorescence intensity (MFI) normalized to the MFI of the isotype control, n = 10. b Histograms of a representative experiment are shown. c M1 macrophages differentiated in the presence or absence of ibrutinib (0.3 µM) as described above were seeded on top of a thick layer of Matrigel in the upper Transwell chamber. The lower chamber was filled with medium with CCL5. Migration was quantified after 3 days. Results are shown as the percentage of migrated macrophages and representative images are shown in the right panel. Representatives images from the top and inside of matrigel are shown. d M1 macrophages were obtained as described above. M2 macrophages were obtained by culturing monocytes from healthy donors with M-CSF (50 ng/ml) for 7 days and either IL-4 (20 ng/ml) or IL-10 (10 ng/ml) were added during the last 2 days of culture. Ibru-M1 macrophages were treated with ibrutinib during the last 2 days of culture. At day 7, the medium was removed and fresh medium without cytokine was added. Then TNF-α and IL-10 was evaluated after 24 h of culture by ELISA. Bars show mean ± SEM. e M1 macrophages differentiated in the presence or absence of ibrutinib (0.3 µM) as described above. At day 7, glucose and lactate concentration in the supernatant was evaluated by using commercial kits. Glucose consumption was calculated as the percentage of glucose in the culture at day 7 relative to the initial glucose concentration in the medium. f M1 macrophages were differentiated in the presence or absence of ibrutinib as described before and stimulated with irradiated Mtb-FITC (MOI equivalent to 5) for 2 h, then cells were trypsinized and the percentage of FITC⁺ macrophages was analyzed by flow cytometry. Results are shown as the percentage of FITC⁺ macrophages, n = 10. g, h Monocytes from healthy donors were differentiated into M1 macrophages and were infected with the red fluorescent protein (RFP) expressing M. tuberculosis strain at an MOI of 5 during 2 h at 37 °C. Thereafter, ibrutinib at 0.3 µM or vehicle was added. After 48 h, the glass coverslips were fixed with PFA 4% and stained with BODIPY 493/503 (Life Technologies). Finally, slides were mounted and visualized with a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan) equipped with a Plapon ×60/NA1.42 objective and then analyzed with the software ImageJ-Fiji. g Quantification of the occupied area with RFP-M. tuberculosis (expressed as Raw Integrated Density) per cell in z-stacks from confocal laser scanning microscopic images. Individual cells were defined by BODIPY-stained cellular membranes that allow us to define the regions of interest for quantification. In all, 80–100 cells of random fields per condition were analyzed. h Representative microphotographs are shown. i, j Purified γδ T cells were stimulated with irradiated Mtb (MOI equivalent to 5) in the presence or absence of ibrutinib. After 24 h, CD69 expression and IFN-γ production were evaluated by flow cytometry and ELISA, respectively. i Results are shown as the percentage of γδ T cells expressing CD69. j IFN-γ concentration in the culture supernatant evaluated by ELISA. *p < 0.05, Wilcoxon matched-pairs signed rank test. ^#p < 0.05, Kruskal–Wallis test, followed by Dunn’s multiple comparisons test