Effects of ibrutinib on macrophage-polarization and on the immune-response against Mycobacterium tuberculosis

COLADO A; KVIATCOVSKY D; GENOULA M; ALMEJÚN MB; PODAZA E; RISNIK D; ELÍAS E; COUGOULE C; MARIDONNEAU-PARINI I; BEZARES F; FERNANDEZ GRECCO H; GIORNADO, M; SASIAIN MC; GAMBERALE R; BALBOA L; BORGE, M

Congreso; International Workshop on Chronic Lymphocytic Leukemia (iwCLL); 2017

Introduction: The Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has been approved for first-line treatment of chronic lymphocytic leukemia (CLL) patients by the FDA. Although durable responses are usually observed with this agent, complete responses are not common and patients have to remain under treatment for long periods. Besides its effects on leukemic Bcells,ibrutinib also affects other cell populations from the tumor microenvironment and the immune system such as T cells, NK cells and macrophages (H. E. Kohrt et al., 2014,J.Dubovsky et al., 2013, M. Borge et al., 2015, S. Fiorcari et al., 2016).Macrophages are central players of the innate immune response against different pathogens including fungi, extracellular bacteria and in particular against intracellular bacteria like Mycobacterium tuberculosis (Mtb), the causing agent of tuberculosis. The World Health Organization (WHO) estimates that one-third of the world's population is infected with Mtb.Importantly, the incidence rate of this disease is highly variable among different countries. For instance, South American countries such as Argentina and Brazil, where ibrutinib was recently approved, have incidence rates of tuberculosis seven and twelve times higher than USA respectively.Considering the key role of macrophages, in particular the M1 profile, in the immune response against Mtb, in this work we proposed to explore the effect of ibrutinib on macrophage polarization and on the effector response to Mtb.Methods: Macrophages were differentiated from healthy donor's or CLL patient's monocytes.M1 polarization was performed with GM-CSF (50 ng/ml) plus IFN-ɣ (10 ng/ml) and IL-4 induced or IL10-induced- M2 polarization with M-CSF (50 ng/ml) plus IL-4 (20 ng/ml) or IL-10 (10 ng/ml).Ibrutinib or the vehicle (DMSO) was added during the last 2 days of culture. HLA-DR, MR, CD86, CD16, CD163, CD14 and MerTK expression was assessed by flow cytometry. TNF-α, IL-8 and IL-10 secretion was measured by ELISA after macrophage stimulation with irradiatedMtb, LPS or Pam3CK for 24 h. Phagocytosis of Mtb-FITC by macrophages was evaluated by flow cytometry. Bacillary loads were determined by colony-forming units assay. Migration in response to CCL5 was measured in matrigel chambers. Statistical significance was determined using the non parametric Friedman test followed by the Dunn?s post test.Results: First we evaluate the effect of ibrutinib on macrophage polarization and found that ibrutinib impaired M1, but not M2, polarization by downregulating CD86 and upregulating MR, CD16 and CD14 expression (Figure 1A-D) (n=8 p˂0.05 control vs ibru), while HLA-DR, CD163 and MerTK expression was not modified. Additionally, ibrutinib enhanced macrophage migration in Matrigel (n=3, p˂0.05), a characteristic associated with the M2 profile (Figure 1E).We also evaluated the effect of ibrutinib on cytokine secretion by Mtb-stimulated macrophages from healthy donors. We were particularly interested in TNF-α production given that the anti-TNF-α therapy used in chronic inflammatory diseases is associated with tuberculosis reactivation. We found that ibrutinib significantly reduced the release of TNF-α, IL-10 and IL-8, being the production of TNF-α severely affected even at low concentrations of the drug, whileIL-10 and IL-8 secretion was only affected at 3 μM (n=10, p˂0.05) (Figure 1F-H). Same results were observed with macrophages from CLL patients (n=6, p