BTK Inhibitors Impair Platelet-Mediated Antifungal Activity

Abstract

In recent years, the introduction of new drugs targeting Bruton's tyrosine kinase (BTK) has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell neoplasms. Although these small molecules were initially considered less immunosuppressive than chemoimmunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis (IA). BTK represents a crucial molecule in several signaling pathways depending on different immune receptors. Based on a variety of specific off-target effects on innate immunity, namely on neutrophils, monocytes, pulmonary macrophages, and nurse-like cells, ibrutinib has been proposed as a new host factor for the definition of probable invasive pulmonary mold disease. The role of platelets in the control of fungal growth, through granule-dependent mechanisms, was described in vitro almost two decades ago and is, so far, neglected by experts in the field of clinical management of IA. In the present study, we confirm the antifungal role of platelets, and we show, for the first time, that the exposure to BTK inhibitors impairs several immune functions of platelets in response to Aspergillus fumigatus, i.e., the ability to adhere to conidia, activation (as indicated by reduced expression of P-selectin), and direct killing activity. In conclusion, our experimental data suggest that antiplatelet effects of BTK inhibitors may contribute to an increased risk for IA in CLL patients.

Keywords: Aspergillus; BTK inhibitors; CLL; acalabrutinib; ibrutinib; invasive fungal infections; molds; platelets.

Figure 1

Experimental workflow. Platelet-rich plasma (PRP) from healthy donors and treatment-naïve CLL patients was treated with ibrutinib or acalabrutinib or vehicle (DMSO) for 60 min at 37 °C. Platelet adhesion to conidia was assessed by a spectrophotometric method. Surface expression of CD62P (P-selectin) was evaluated by flow cytometry at baseline and following stimulation by A. fumigatus conidia. Platelet-induced hyphal damage was measured by performing a colorimetric assay with XTT. Platelet-mediated antifungal activity was also evaluated in CLL patients before and after ibrutinib administration by cytofluorimetric analysis of P-selectin expression in response to A. fumigatus conidia.

Figure 2

Ibrutinib inhibits adhesion of platelets to conidia. Platelets, treated or not treated with three different concentrations of ibrutinib (0.2 μM, 0.5 μM, and 1 μM), were incubated with A. fumigatus conidia, at an effector-to-target (E:T) ratio of 100:1 for 30 min at 37 °C, and then centrifuged at low intensity. Then, the OD₇₀₀ of the supernatant was determined by spectrophotometer, and the percentage of platelet adhesion was calculated in relation to the OD₇₀₀ of platelets and conidia alone (** p < 0.01; *** p < 0.001; **** p < 0.0001). HS, healthy subjects; PTs, patients; CTRL, control; IBRU, ibrutinib.

Figure 3

In vitro effects of ibrutinib and acalabrutinib on platelets’ degranulation in response to A. fumigatus conidia. Platelet-rich plasma (PRP) from healthy volunteers (A) and treatment-naïve CLL patients (B) was treated with 1 μM ibrutinib (IBRU), acalabrutinib (ACALA), or vehicle (DMSO) for 1 h at 37 °C and stimulated or not stimulated with A. fumigatus conidia at an effector-to-target (E:T) ratio of 100:1 for 30, 60, 90, 120, 180, or 270 min. Platelet activation was detected by flow cytometry, labeling samples with PE-conjugated anti-CD42b antibody, a surface marker constitutively expressed on platelets, and with an FITC-conjugated anti-CD62P/P-selectin antibody, a marker of alpha granule secretion. Results are reported as percentages of CD62b expression normalized on DMSO-treated unstimulated platelets (** p < 0.01; *** p < 0.001).

Figure 4

In vitro effects of ibrutinib and acalabrutinib on platelet-mediated hyphal damage. Platelet-rich plasma (PRP) from healthy volunteers (A) and treatment-naïve CLL patients (B) was treated with 1 μM ibrutinib (IBRU), acalabrutinib (ACALA), or vehicle (DMSO) for 1 h at 37 °C. A. fumigatus conidia were incubated for 16 h at 37 °C in RPMI medium plus 1% sodium pyruvate to produce hyphae with or without platelets, at an effector-to-target (E:T) ratio of 100:1. For measurement of hyphal metabolic activity, XTT salt plus 40 μg/mL coenzyme Q was added. Absorbance was determined at 450 nm using an enzyme-linked immunosorbent assay plate reader, and antifungal activity was calculated as the percentage of hyphal damage equal to [1 − (X/C)] × 100, where X is the optical density of test well and C is the optical density of control wells with hyphae only (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

Platelet-mediated antifungal activity in CLL patients (PT) under ibrutinib. Bar (A) and line graph (B) showing P-selectin expression in samples collected before starting ibrutinib and during the course of treatment (* p < 0.05; ** p < 0.01).

Figure 6

BTK inhibitors (BTKis) suppress (×) antifungal immune responses (⊥) mediated by macrophages, nurse-like cells (NLCs), monocytes, neutrophils, and platelets. The fungal cell wall contains polysaccharides and lipid moieties that elicit an immune response with a strong production of cytokines, comprising tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), IL-6, and IL-8. The recognition of fungi by innate immune cells relies on the interaction between pathogen-associated molecular patterns (PAMPs, i.e., β-glucans, chitins, and mannans) and different pattern recognition receptors (PRRs). The typical PRRs for A. fumigatus encompass β-glucan receptor (Dectin-1), complement receptor 3 (CR3), triggering receptor expressed on myeloid cells-1 (TREM-1), and Toll-like receptors (TLRs). BTK is crucially involved in the transmission of multiple signaling cascades activated by PRRs and, therefore, can be considered a ‘guardian’ of the innate immunity. Ibrutinib and acalabrutinib compromise the ability of neutrophils, monocyte/macrophage populations, and platelets to counteract the fungal growth. Of note, acalabrutinib, unlike ibrutinib, has not yet been associated with an increased risk for invasive fungal infections (IFIs) in treated patients, nor have detrimental effects on antifungal innate immune responses been described in vivo so far. It is conceivable that the presence of functional TEC (not targeted by acalabrutinib) may partially compensate for nonfunctional BTK in non-B cells, as suggested for patients with X-linked agammaglobulinemia (XLA), showing unremarkable rates of IFIs. The combined inhibition of BTK and TEC (both targeted by ibrutinib) is expected to fully suppress BTK/TEC-dependent inflammatory pathways, thus leading to an augmented susceptibility to IFIs. FcγR, Fc-gamma receptor; ROS, radical oxygen species; NETs, neutrophil extracellular traps; PSGL-1, P-selectin glycoprotein ligand 1; ↑, increase; ↓, decrease.