Alteration of anti-Aspergillus innate immune responses by Bruton’s tyrosine kinase inhibitors

Author:

Marion Blaize1,2, Guillaume Thizy2, Arnaud Fekkar1,2

Author address:

1APHP.Sorbonne Université, Hôpital La Pitié Salpêtrière, Parasitologie Mycologie, Paris, France, Paris, France
2Sorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses, CIMI-Paris, Paris, France

Abstract:

Objectives:
Neutrophils and macrophages are innate immune cells that play a central role against Aspergillus. Development of Bruton’s tyrosine kinase (Btk) inhibitors (BTKi), which block proliferation, and survival of B-lymphocytes has improved prognosis of chronic lymphocytic leukemia. Ibrutinib, the first labelled BTKi, has a broad spectrum of activity on different kinases and cardiac, hemorrhagic and infectious side effects have been described, in particular invasive aspergillosis. New generation drugs with fewer side effects due to greater selectivity on Btk have been developed. We and others have previously pointed out some defects in the innate response to Aspergillus when neutrophils are exposed to ibrutinib. However, no data are available regarding the impact of second- and third-generation BTKi on innate immunity. We present an original approach combining flow cytometry and fluorescence video-microscopy to analyze the impact of three BTKi on neutrophil and macrophage responses against Aspergillus.
Methods:
Blood samples from healthy donors were incubated with different BTKi (ibrutinib, acalabrutinib and zanubrutinib) for 15 minutes. Whole blood stimulation was performed with Aspergillus fumigatus germinating conidia. Reactive oxygen species (ROS) production and neutrophil surface expression of CD11b and neutrophil’s phagocytic capacity of dormant conidia were monitored by flow cytometry. The microbicidal activity of neutrophils and monocyte-derived macrophages (MDM) was assessed by an original approach based on video-microscopy. Purified neutrophils were incubated with A. fumigatus Ds-Red germinating conidia overnight and video-microscopy was used to quantify cells/fungus interactions (engulfment) and fungal killing. MDM were incubated with A. fumigatus Ds-Red resting conidia overnight and video-microscopy allowed to quantify phagocytosis and inhibition of Aspergillus growth.
Results:
The results indicate several notable defects in the host responses to Aspergillus. Neutrophil surface expression of CD11b integrin following stimulation by germinating conidia was decreased by around 20%-30% by all inhibitors. ROS production was also reduced by around 50% in the presence of ibrutinib, zanubrutinib and acalabrutinib, the latter molecule having the least impact on ROS production. Phagocytosis of resting conidia by neutrophils decreased from 40% to 20% when neutrophils were exposed to acalabrutinib, and to 12% and 11% with ibrutinib and zanubrutinib, respectively.
The video-microscopy approach reveals important defects in the Aspergillus uptake by immune cells. While no change in conidial engulfment was observed, killing of germinating conidia was clearly affected by the different BTKi tested, with a up to 50% decrease of killing when neutrophils were exposed to zanubrutinib.
Phagocytosis of resting Aspergillus conidia by MDM was also significantly impaired after exposure to the different BTKi. Finally, analyze of the proportion of internalized conidia that were able to start germination after 12 hours of MDM/fungus interaction show inhibition of 75% of conidial germination by control MDM while treated MDM inhibit only 63%, 54% and 52% after exposure to acalabrutinib, ibrutinib and zanubrutinib respectively.
Conclusions:
Our results indicate that new BTK inhibitors lead to defects in the neutrophils and macrophages responses to Aspergillus. These preliminary data need to be confirmed by enrolling patients treated by these new BTKi. It seems important to follow patients treated with new generation BTKi for the risk of aspergillosis.

Abstract Number: 59

Conference Year: 2024

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