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 5

BTK mediates p40phox and RAC2 activation in human neutrophils.

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BTK mediates p40phox and RAC2 activation in human neutrophils. (A–C) Imm...
(A–C) Immunoblot analysis of p40phox phosphorylation (at T154) in human neutrophils upon stimulation with serum-opsonized heat-killed Aspergillus conidia at the indicated time points. Representative immunoblot images (top panels) and quantified pixel density values (lower panels) are shown. p40phox phosphorylation is shown in healthy donor neutrophils treated with vehicle or ibrutinib (A), in neutrophils from patients with lymphoma, isolated before or at day 3 after initiation of treatment with ibrutinib or acalabrutinib (B), and in neutrophils isolated from healthy donors or patients with XLA (C). (D) Representative immunoblot images depicting active RAC2-GTP and total RAC2. Left: pull-down was performed using unstimulated healthy donor neutrophil lysates in the presence of GDP (negative control) and GTPγS (positive control). Right: pull-down was performed using healthy donor neutrophil lysates, following neutrophil treatment with vehicle, ibrutinib, or buffer (Hank’s balanced salt solution: HBSS) and stimulation with serum-opsonized zymosan. (E) Quantification of the ratio of active RAC2-GTP relative to total RAC2, normalized to the vehicle-treated neutrophils. Each dot depicts an individual healthy donor or patient. Box and whisker plots depict values ranging from minimum to maximum (C). Bars depict mean ± SD. Ibrutinib concentration, 2.5 μM. BTKi, BTK inhibitor. *P<0.05, **P<0.01, ***P<0.001, determined using 2-sided paired t test (A and B), 2-sided Wilcoxon test (B), 2 sided unpaired t test (C) or 2-sided Mann-Whitney U test (C), or 2-sided Welch’s t test (E). Grubb’s outlier test applied with 1 outlier excluded (C: 20 min timepoint).