Fig. 1: Ibrutinib impairs macrophage-mediated response against Mycobacterium tuberculosis.

Macrophages were obtained by culturing monocytes from healthy donors (HD) or CLL patients for 5 days in RPMI 10% FCS in the presence of M-CSF (50 ng/ml). a–c HD-macrophages were stimulated with irradiated Mtb (MOI equivalent to 2) in the presence or absence of ibrutinib (Ibru) and after 24 h TNF-α, IL-8, and IL-10 secretion was measured by ELISA in culture supernatants. Bars represent mean ± SEM of cytokine concentration in control (ct.) cultures (white bars) or Mtb-stimulated cultures (gray bars). n = 10, *p < 0.05, Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. d Phosphorylation of p65 (p-p65) was evaluated by western blot in HD macrophages after 15 min of stimulation with Mtb in the presence or absence of ibrutinib. Bands on the immunoblots were quantified using the ImageJ software (NIH Image). Results are shown as the mean ± SEM of the ratio p-p65/β-actin in arbitrary units (A.U.). n = 6, *p < 0.05, Friedman test, followed by Dunn’s multiple comparisons test. e HD macrophages were stimulated with LPS (100 ng/ml) or Pam3CSK4 (100 ng/ml) in the presence or absence of ibrutinib for 24 h and TNF-α production was measured in culture supernatants by ELISA. n = 10, *p < 0.05, Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. f CLL macrophages were stimulated with irradiated Mtb (MOI equivalent to 2), LPS (100 ng/ml), or Pam3CSK4 (100 ng/ml) in the presence or absence of ibrutinib for 24 h and TNF-α production was measured by ELISA in culture supernatants. n = 7, *p < 0.05, Kruskal–Wallis test, followed by Dunn’s multiple comparisons test