BTK drives neutrophil activation for sterilizing antifungal immunity
Jigar V. Desai, … , Tobias M. Hohl, Michail S. Lionakis
Jigar V. Desai, … , Tobias M. Hohl, Michail S. Lionakis
Published May 2, 2024
Citation Information: J Clin Invest. 2024;134(12):e176142. https://doi.org/10.1172/JCI176142.
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Research Article Immunology Infectious disease

BTK drives neutrophil activation for sterilizing antifungal immunity

Abstract

We describe a previously unappreciated role for Bruton’s tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, patients who were treated with BTKi, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in patients who are susceptible.

Authors

Jigar V. Desai, Marissa A. Zarakas, Andrew L. Wishart, Mark Roschewski, Mariano A. Aufiero, Agnes Donkò, Gustaf Wigerblad, Neta Shlezinger, Markus Plate, Matthew R. James, Jean K. Lim, Gulbu Uzel, Jenna R.E. Bergerson, Ivan Fuss, Robert A. Cramer, Luis M. Franco, Emily S. Clark, Wasif N. Khan, Daisuke Yamanaka, Georgios Chamilos, Jamel El-Benna, Mariana J. Kaplan, Louis M. Staudt, Thomas L. Leto, Steven M. Holland, Wyndham H. Wilson, Tobias M. Hohl, Michail S. Lionakis

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Figure 4

BTK promotes neutrophil hyphal damage, the oxidative burst, and primary granule release.

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BTK promotes neutrophil hyphal damage, the oxidative burst, and primary ...
(A) A. fumigatus hyphal damage induced by neutrophils from healthy donors or patients with XLA (effector:target ratio, 8:1) (n = 9). (B and C) Representative histograms (B) and MFI summary data (C) depicting dihydrorhodamine 123 oxidation to rhodamine in neutrophils from healthy donors or patients with XLA stimulated as indicated (n = 9–10). (D and E) Luminol-based assay of ROS production. Temporal trace of chemiluminescence RLU (D), and AUC for RLU (E), when neutrophils from healthy donors or patients with XLA were stimulated as indicated (n = 7–12). (F) Neutrophils of healthy donors or patients with XLA were coincubated with A. fumigatus hyphae (effector:target ratio, 8:1) and the indicated granule components were analyzed via ELISA in the supernatants (n = 8–22). Each dot in panels A, C, E, and F represents an individual healthy donor or a patient with XLA. (G) A. fumigatus hyphal damage induced by bone marrow neutrophils isolated from WT or Btk–/– mice (effector:target ratio, 32:1) (n = 6). (H) MFI summary data that depict dihydrorhodamine 123 oxidation to rhodamine in neutrophils isolated from the Aspergillus-infected lung of Btk+/+ or Btk–/– mice at day 2 after infection, stimulated as indicated (n = 4). (I) Bone marrow neutrophils were coincubated with A. fumigatus hyphae at a 32:1 effector:target ratio and myeloperoxidase (MPO) was analyzed via ELISA in the supernatants (n = 6). Each dot in panels G and H represents an individual mouse, and each dot in I represents 1–3 technical replicates from 9 individual mice. Box and whisker plots depict values ranging from minimum to maximum (A, C, and E–I). MPO: myeloperoxidase; MMP-9: Matrix metalloproteinase-9; PMA, phorbol-12-myristate-13-acetate; Af: Aspergillus fumigatus; HK Af: heat-killed Aspergillus fumigatus; XLA: X-linked agammaglobulinemia. *P<0.05, **P<0.01, ***P<0.001 using 2 sided unpaired t test (A, C, and F–I), 2-sided or Mann-Whitney U-test (C and E).