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 2

BTK is activated in human neutrophils upon fungal exposure, and pharmacologic BTK inhibition impairs antifungal effector functions.

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BTK is activated in human neutrophils upon fungal exposure, and pharmaco...
(A) Transcript levels of BTK in human neutrophils. Also shown are CYBB and CD8A as positive and negative control genes, respectively. Data sourced from GSE145033 (49, 50). (B and C) Representative FACS histograms (B) and geometric mean fluorescence intensity (MFI) summary data (C) for phosphorylated BTK in healthy donor neutrophils at baseline and at the indicated time points after stimulation with serum-opsonized, heat-killed (HK) A. fumigatus (Af) conidia (n = 5). (D) A. fumigatus hyphal damage induced by vehicle- or ibrutinib-treated neutrophils at an effector:target ratio of 8:1 (n = 6). (E and F) Representative histograms (E) and MFI summary data (F) that depict dihydrorhodamine 123 oxidation to rhodamine, in vehicle- or ibrutinib-treated healthy donor neutrophils stimulated as indicated (n = 6). (G and H) Luminol-amplified chemiluminescence. (G) Temporal trace of chemiluminescence-based reactive oxygen species (ROS) (in relative light units: RLU) of vehicle- or ibrutinib-treated healthy donor neutrophils stimulated as indicated. (H) AUC summary data for luminol-amplified chemiluminescence RLU shown in G (n = 9). (I) Vehicle- or ibrutinib-treated healthy donor neutrophils were coincubated with A fumigatus hyphae at an 8:1 effector:target ratio and the indicated granule components were analyzed via ELISA in the supernatants of neutrophil-hyphal cocultures (n = 4). Each dot represents an individual healthy donor. Quantitative data are means ± SEM (C). Box and whisker plots depict values ranging from minimum to maximum (A and F). Ibrutinib concentration, 250 nM. FPM, fragments per million; MPO, myeloperoxidase; MMP-9, Matrix metalloproteinase-9; PMA, phorbol-12-myristate-13-acetate; Af, Aspergillus fumigatus; HK Af, heat-killed Aspergillus fumigatus. *P<0.05, **P<0.01, ***P<0.001, determined using 1-way ANOVA with Dunnett’s multiple comparisons test (C), or 2-sided paired t test (D, F, H, and I). Grubb’s outlier test applied with 1 outlier excluded (I: MPO).