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Ibrutinib impairs anti-Aspergillus responses
dictable decrease in ROS in response to germinating coni- dia, but also decreased killing activity against growing Aspergillus, in a manner similar to that observed with ibru- tinib (Figure 6E, F and data not shown). Interestingly, DPI had no effect on the engulfment process, which suggests that the inhibitory effect of ibrutinib is not caused by the sole inhibition of ROS production.
Discussion
Ibrutinib, an irreversible BTK inhibitor, displays signifi- cant clinical efficacy in some lymphoid malignancies.2 Ibrutinib has, however, recently been associated with the occurrence of invasive fungal infections, in particular inva- sive aspergillosis.3-5 Intriguingly, patients with XLA do not experience aspergillosis and patients with lymphoid malignancies are usually considered to be at low risk of invasive aspergillosis. Consequently, the mechanism behind the emergence of invasive aspergillosis among patients receiving ibrutinib has remained unclear. The association of ibrutinib and fungal infections, in particular invasive aspergillosis,3-5 suggested that the drug could have a detrimental effect on antifungal immune responses.17
Because neutrophils are the cornerstone in the fight against fungi, the impact of ibrutinib on neutrophil func- tions had to be evaluated. Neutrophils act against Aspergillus through different mechanisms, depending on the stage of life of the fungus. Inhibition of the growth of resting conidia involves non-oxidative pathways and CD11b/CD18 recognition while hyphae are destroyed by oxidative derivatives after recognition by Fc-gamma receptor or CLR.18,19
Analysis of CD11b, which like Dectin-1 is a very important receptor for β-glucan,20 showed conflicting results. An unpaired comparison between 17 samples obtained before treatment and 17 samples after approxi- mately 1 month of ibrutinib indicated that basal levels were reduced after initiating ibrutinib therapy. This result was further confirmed in vitro. However, paired analysis on eight longitudinally followed patients was inconclu- sive. The reason for this discrepancy is unclear. It might be due to an inadequate cohort size or to the patients’ heterogeneity in terms of disease stage and treatment response. Interestingly, both in vivo and in vitro analyses showed that ibrutinib had no effect on CD11b surface expression in response to LPS, which suggests that the defect may be specific to the antifungal response. The C- type lectin receptor of β-glucan Dectin-1 is considered to be an essential component of the antifungal immune response. In human neutrophils Dectin-1 neutralization by monoclonal antibodies leads to an impairment of binding and phagocytosis of zymosan and diminished ROS production21 although contradictory results have been published.20 We found a not statistically significant defect in Dectin-1 surface expression during ibrutinib therapy. Collectively, our results suggest that ibrutinib may alter CD11b surface expression on neutrophils from treated patients but has no effect on other important immune receptors and has little effect on their upregula- tion after Aspergillus and LPS stimuli.
ROS production is a major mechanism to kill fungi7,8 as exemplified by the phenotype of patients with chronic granulomatous disease. Neutrophils of these patients dis- play a profound defect in ROS production caused by
mutations in components of the NADPH oxidase22 and therefore are unable to kill Aspergillus hyphae.19 Mangla et al. showed that neutrophils from X-linked immunodefi- cient mice lacking functional Btk had defective produc- tion of both ROS and nitric oxide after LPS challenge.23 In agreement with these observations, a recent study also found that superoxide production in neutrophils stimulat- ed with fMLP was reduced in Btk-/- mice.24 After 1 month of ibrutinib treatment, we found that neutrophil ROS decreased by approximately 50% in the presence of Aspergillus germinating conidia. A similar alteration in ROS production was also observed when ibrutinib was added to blood from healthy donors, which indicates that the impact of ibrutinib is prompt and seems to rule out an effect occurring during granulopoiesis. In contradiction with our results, Stadler et al. found no difference in ROS production by neutrophils challenged with zymosan or LPS in patients treated with ibrutinib.11 It should however be noted that these results were obtained from a cohort of only six patients, for whom the duration and posology of ibrutinib therapy were not reported. Moreover, zymosan is a yeast extract containing β-glucan, the struc- ture of which differs from that of Aspergillus. Since CD11b is important for neutrophil ROS production,25 one may hypothesize that reduced CD11b expression after ibrutinib exposure may affect ROS, although this requires further investigation.
In human neutrophils, it has been shown that fMLP- induced IL-8 release is dependent on ROS production.26 IL-8 is a major neutrophil chemoattractant and its levels have been found to be elevated in the serum and bron- choalveolar lavage of patients with invasive aspergillo- sis.27 IL-8 production by neutrophils was reduced in sam- ples from ibrutinib-treated patients upon Aspergillus stim- ulation. By contrast, the production of other cytokines did not change upon stimulation or during the course of ibrutinib therapy. Whether or not defective IL-8 produc- tion may result in insufficient neutrophil recruitment to the sites of Aspergillus infection deserves further investiga- tion, including the use of animal models.
While ibrutinib did not seem to impair neutrophil chemotaxis, video-microscopy revealed that it reduced the cells’ ability to interact closely with and engulf Aspergillus. The molecular mechanism by which ibrutinib hinders these interactions remains to be determined. More striking was the dramatic decrease in the ability of neutrophils to kill Aspergillus when exposed to ibrutinib. Altogether, given that the oxidative burst is the major mechanism of killing hyphae,18 it is very likely that ibru- tinib impairs neutrophil killing at least in part through inhibition of ROS. We observed that in vitro exposure of neutrophils to the NADPH oxidase inhibitor DPI recapit- ulated the effect of ibrutinib except it did not impair Aspergillus engulfment by neutrophils, which suggests that the effect of ibrutinib on neutrophils extends beyond that of ROS inhibition and involves different pathways.
Collectively, our results show that ibrutinib induces multiple functional defects in neutrophils, which result in their inability to kill Aspergillus, and provide a first clue to explain clinical cases. One caveat of our study is that it was not designed to determine whether the effect of ibru- tinib is caused by the inhibition of BTK itself. Essentially known for its key role in B cells, experimental evidence suggests that BTK is functional in neutrophils. In a Btk-
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