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D. Blez et al.
deficient mouse model, Fiedler et al. showed that Btk was important during granulopoiesis and that neutrophils from Btk-/- mice lacked granule components important for their antimicrobial activity, such as elastase, lactoferrin and myeloperoxidase.10 Btk is involved in the activation of the PLCγ2, AKT and p38MAPK pathways in neu- trophils after stimulation.24 Btk is also involved in phos- phorylation of Myd88 and NFκB activation after TLR2 or TLR4 engagement.28,29 Lionnakis et al. reported that Btk-/- mice died after Aspergillus challenge whereas wildtype animals did not.4 However, not all experimental evidence agrees with such a role for Btk. Indeed, Btk was found by Honda et al. to be a critical gatekeeper of neutrophil responses, preventing excessive inflammation through inhibition of ROS.30 More recently Cavaliere et al. con- cluded that monocyte and neutrophil maturation and function were unaffected in XLA patients.31 Another lim- itation of our in vitro experiments is that we, as others,17 used higher concentrations of ibrutinib than those observed in patients32 to compensate for the very short time of exposure. However, several neutrophil functions, such as an increase in CD11b expression after LPS chal- lenge, were unchanged, suggesting that the concentra- tions used did not lead to a global impairment of the neu- trophils’ cellular processes.
XLA patients are not particularly susceptible to fungal infections, unless one considers that alternative mecha- nisms may develop over time to compensate for BTK deficiency in humans. At present, whether neutrophil defects are caused by the sole inhibition of BTK by ibru- tinib thus remains an open question. Like most kinase inhibitors, ibrutinib is not highly specific, which raises the hypothesis that impairment of antifungal activity may be caused by inhibition of other targets. Further work is required to unravel the precise molecular mecha- nisms responsible for the observed neutrophil defects. The development of a murine model would provide inter-
esting information on the antifungal response dynamics in vivo, e.g., whether the impact on CD11b alters the neu- trophils’ ability to reach infected tissues. A comparison with other BTK inhibitors, in particular acalabrutinib, which is more selective than ibrutinib, might help to explore the hypothesis of an off-target effect. Finally, it should be highlighted that despite these experimental results, invasive aspergillosis remains a relatively rare complication. This suggests that ibrutinib exposure is probably not sufficient by itself in most cases and that additional environmental and host factors, e.g., TLR or Dectin polymorphisms, the amount of conidia exposure, acquired immune defects related to the underlying dis- ease or previous therapies, are required to enable the development of a clinical infection.
In conclusion, our study, which demonstrates that expo- sure to ibrutinib impairs the anti-Aspergillus responses of neutrophils both in vitro and in vivo, provides the first step in the road to understanding the relationship between invasive fungal infections and treatment with ibrutinib. The emer- gence of invasive aspergillosis in this population may be due to neutrophil defects in ROS production in response to fungi, inability of neutrophils to attach firmly to hyphae, and marked impairment in the neutrophils’ capacity to kill fungi. Further work is required to unravel the exact mecha- nisms underlying this effect, in particular whether it is caused by the inhibition of BTK itself in neutrophils or by an off-target inhibition of other kinases in neutrophils and/or other cell types in the microenvironment.
Acknowledgments
We thank all patients and express our gratitude to healthcare providers for their assistance in the inclusion of patients.
This work was supported by a grant from The Janssen Company and grants from “Fondation pour la Recherche Médicale - Equipe Labelisée” and from “Agence Nationale de la Recherche”, project CMOS 2015 (ANR-EMMA-050).
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