Research ArticleImmunologyInfectious disease Open Access | 10.1172/jci.insight.176162

Diego A. Vargas-Blanco,1,2 Olivia W. Hepworth,1,2 Kyle J. Basham,1 Patricia Simaku,1 Arianne J. Crossen,1 Kyle D. Timmer,1 Alex Hopke,2,3,4 Hannah Brown Harding,1,2 Steven R. Vandal,5 Kirstine N. Jensen,1,2 Daniel J. Floyd,1 Jennifer L. Reedy,1,2 Christopher Reardon,1 Michael K. Mansour,1,2 Rebecca A. Ward,1 Daniel Irimia,2,3,4 Jeremy S. Abramson,6 and Jatin M. Vyas1,2

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

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1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

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1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

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1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

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1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

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1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

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1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Hopke, A. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Brown Harding, H. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Vandal, S. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Jensen, K. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Floyd, D. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Reedy, J. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Reardon, C. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Mansour, M. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Ward, R. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Irimia, D. in: JCI | PubMed | Google Scholar |

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Abramson, J. in: JCI | PubMed | Google Scholar

1Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

2Harvard Medical School, Boston, Massachusetts, USA.

3BioMEMS Resource Center, Massachusetts General Hospital, Boston, Massachusetts, USA.

4Shriners Hospital for Children, Boston, Massachusetts, USA.

5Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

6Center for Lymphoma, Mass General Cancer Center, Boston, Massachusetts, USA.

Address correspondence to: Jatin M. Vyas, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114, USA. Email: jvyas@mgh.harvard.edu.

Authorship note: DAVB and OWH are co–first authors.

Find articles by Vyas, J. in: JCI | PubMed | Google Scholar |

Authorship note: DAVB and OWH are co–first authors.

Published May 7, 2024 - More info

Published in Volume 9, Issue 12 on June 24, 2024
JCI Insight. 2024;9(12):e176162. https://doi.org/10.1172/jci.insight.176162.
© 2024 Vargas-Blanco et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published May 7, 2024 - Version history
Received: September 25, 2023; Accepted: April 23, 2024 View PDF Abstract

Inhibition of Bruton’s tyrosine kinase (BTK) through covalent modifications of its active site (e.g., ibrutinib [IBT]) is a preferred treatment for multiple B cell malignancies. However, IBT-treated patients are more susceptible to invasive fungal infections, although the mechanism is poorly understood. Neutrophils are the primary line of defense against these infections; therefore, we examined the effect of IBT on primary human neutrophil effector activity against Aspergillus fumigatus. IBT significantly impaired the ability of neutrophils to kill A. fumigatus and potently inhibited reactive oxygen species (ROS) production, chemotaxis, and phagocytosis. Importantly, exogenous TNF-α fully compensated for defects imposed by IBT and newer-generation BTK inhibitors and restored the ability of neutrophils to contain A. fumigatus hyphal growth. Blocking TNF-α did not affect ROS production in healthy neutrophils but prevented exogenous TNF-α from rescuing the phenotype of IBT-treated neutrophils. The restorative capacity of TNF-α was independent of transcription. Moreover, the addition of TNF-α immediately rescued ROS production in IBT-treated neutrophils, indicating that TNF-α worked through a BTK-independent signaling pathway. Finally, TNF-α restored effector activity of primary neutrophils from patients on IBT therapy. Altogether, our data indicate that TNF-α rescued the antifungal immunity block imposed by inhibition of BTK in primary human neutrophils.

Graphical Abstract Introduction

Invasive fungal infections are dreaded complications for those with compromised immune systems, including patients with cancer (e.g., leukemia, lymphoma) and solid-organ and hematopoietic stem cell transplant recipients. The fungal pathogen Aspergillus spp. causes a spectrum of diseases, including asthma, chronic infection, and invasive disease. Invasive fungal infections carry elevated mortality rates in these high-risk patients, despite the availability of antifungals (1–4), demonstrating the critical role of the innate immune system as the first line of defense against these devastating infections (5, 6).

As the first responders in fungal infections, neutrophils exert antifungal activity through multiple effector functions, including swarming, phagocytosis, and reactive oxygen species (ROS) production. Activation of neutrophil pattern recognition receptors triggers these effector functions and subsequent cytokine secretion. However, a reduced ability to produce neutrophils or neutrophil dysfunction occurs in many immunosuppressed individuals, contributing to an elevated risk of invasive fungal infections, including invasive aspergillosis. Tyrosine kinases are critical to neutrophil effector function in antifungal immunity (7–9). Aspergillus cell wall carbohydrates trigger intracellular signaling cascades and effector functions through spleen tyrosine kinase (Syk) (10, 11). Bruton’s tyrosine kinase (BTK), a kinase downstream of Syk, mediates antifungal response in innate immune cells, including neutrophils (12). While these kinases are critical in antifungal immunity, small-molecule inhibitors targeting these molecules are effective therapeutics for B cell malignancies and chronic graft-versus-host disease (13–16).

Unfortunately, BTK inhibitor therapy amplifies the risk of invasive infections, including fungal pathogens, particularly in dissemination to the central nervous system (CNS) (15, 17–20). Although BTK inhibitors (e.g., acalabrutinib [ABT], ibrutinib [IBT], zanubrutinib [ZBT]) improve outcomes in multiple subtypes of B cell lymphoma and leukemia, BTK, and other Tec protein tyrosine kinases signal diverse cellular processes in immune cell lineages (e.g., macrophages, neutrophils, γδ T cells) (21–24). These BTK inhibitors impair the function of immune cells critical to host defense against invading pathogens through the suppression of proinflammatory cytokines, dampened killing capacity, and blunted ROS production (19, 25–32). Indeed, the irreversible inhibitor of BTK, IBT, quickly reduces BTK phosphorylation at the Tyr551 and Tyr223 sites and has been linked to defects in murine neutrophils when responding to Aspergillus fumigatus (29, 31). The effect of BTK inhibition on neutrophil effector functions remains incompletely understood (33).

Here, we demonstrate the deleterious effect of 3 BTK inhibitors (IBT, ABT, and ZBT) on the antifungal effector functions of human neutrophils, including chemotaxis, phagocytosis, and ROS production. Given that genes related to the TNF signaling pathways were the most differentially expressed in IBT-treated neutrophils, we tested the hypothesis that TNF-α could bypass the block imposed by BTK inhibition. We show that exogenous TNF-α improves BTK inhibitor–associated defects, restoring the neutrophil ability to control A. fumigatus in healthy neutrophils treated with BTK inhibitors as well as in neutrophils from IBT-treated patients. We demonstrate that the restorative effect of exogenous TNF-α occurs via transcription-independent signaling. Taken together, these data indicate that exogenous TNF-α acts as a signaling molecule in neutrophils, rapidly compensating for BTK inhibitor–imposed defects in response to A. fumigatus.

Results

IBT inhibited neutrophil effector activity against A. fumigatus. To evaluate the hypothesis that BTK inhibition of neutrophils affect antifungal immune response against A. fumigatus, we sought to determine the effect of BTK inhibition on neutrophil effector functions including killing, ROS production, phagocytosis, and swarming by neutrophils when challenged with A. fumigatus. Primary human neutrophils treated ex vivo with IBT at a physiologically relevant concentration (19, 34) (0.3 μM) or 10-fold higher or lower concentrations failed to kill A. fumigatus in contrast to neutrophils treated with solvent control (0.1% DMSO), as shown by a resazurin-based metabolic assay (Figure 1A). These data were confirmed by calculating the rate of growth inhibition of A. fumigatus when compared with the A. fumigatus growth alone (Figure 1B). These results demonstrate that IBT-treated neutrophils failed to control A. fumigatus growth as compared with solvent-treated neutrophils.

Figure 1

IBT inhibition dampened human neutrophil effector activity against A. fumigatus. (A) Metabolic activity of A. fumigatus B5233 strain measured using resazurin. Human neutrophils were pretreated for 4 hours (4h) with IBT and stimulated with A. fumigatus (MOI:0.25) for 5h. Data are shown as mean ± SD, n = 3; data are representative of at least 3 independent experiments. (B) Percentages of growth inhibition derived from A using linear regression analysis in a Gompertz fit. Data are shown as 95% CI, n = 3. Ordinary 1-way ANOVA and Tukey’s multiple-comparison test with a single pooled variance demonstrated a P < 0.0001 for all IBT treatments versus DMSO alone. (C and D) Human neutrophils were treated for 4h with IBT or DMSO and then stimulated with 1 mg/mL A. fumigatus B5233 strain heat-killed hyphal elements (C) or 1 μg/mL PMA (D). ROS production was measured by chemiluminescence using lucigenin. Data are shown as mean ± SD, n = 3. (E) Human neutrophils were treated with IBT or DMSO for 4h and incubated with Af488-labeled A. fumigatus B5233 strain (conidia+) swollen spores (MOI: 10). A subset of neutrophils was pretreated with 20 μM of cytochalasin D (Cyto D). The displayed percentage of phagocytic neutrophils (CD45-AF700+CD66b-APC+conidia-AF488+) was estimated based on the total number of viable neutrophils (CD45-AF700+CD66b-APC+). A minimum of 10,000 viable CD66b-APC+ events were recorded. (F–H) Human neutrophils were treated with IBT or DMSO for 4h before coincubation with A. fumigatus B5233 strain. Representative microscopy panels from the swarming assay showing neutrophil swarm formations 200 minutes (min) after coincubation, white circles depict areas seeded with A. fumigatus (F). Area of human neutrophil swarm 200 min after coincubation with A. fumigatus seeded spores (G). Area of fungal growth per cluster on swarming array slides after 16h, normalized to A. fumigatus growth without neutrophils (H). Data are shown as mean ± SD, n = 8. Ordinary 1-way ANOVA and Tukey’s multiple-comparison test with a single pooled variance. *P < 0.05; ***P < 0.001; ****P < 0.0001. For all panels, data are representative of at least 3 independent experiments.

We next examined the effects on ROS production in primary neutrophils using the same doses as above. Consistent with the metabolic activity assay, IBT-treated neutrophils produced less ROS in response to heat-killed A. fumigatus hyphae when compared with DMSO-treated neutrophils (Figure 1C). These BTK inhibitor–induced effects on ROS production were not strain specific, and IBT blocked β-glucan–coated bead–induced (the agonist for Dectin-1 signaling) ROS production (Supplemental Figure 1, A–D; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.176162DS1). As a control, we examined the effect of BTK inhibition on Dectin-1 expression in primary human neutrophils as loss of expression of Dectin-1 could be a trivial explanation for these findings. Dectin-1 expression was not altered in IBT-treated neutrophils (Supplemental Figure 1E). To examine whether these effects blocked all induced ROS production, we stimulated IBT-treated neutrophils with PMA, a NADPH oxidase inducer. PMA in the presence of IBT generated ROS similar to the solvent control (Figure 1D), suggesting that IBT-associated ROS defects were specific to ligands found on A. fumigatus. We examined intracellular ROS production to determine if this process was also sensitive to BTK inhibition. IBT potently reduced the amount of intracellular ROS as determined by flow cytometry (Supplemental Figure 2). These data indicate that IBT blocked both extracellular and intracellular ROS production.

Since pathogen-associated molecular pattern molecules can trigger an increase of neutrophilic phagocytic activity (35), we sought to determine whether BTK inhibitor effect included phagocytosis. We measured neutrophil phagocytosis of A. fumigatus by flow cytometry using Alexa Fluor 488–labeled (AF488-labeled) conidia. Neutrophils were gated as the double positive CD45+CD66b+ subpopulation, and evidence of phagocytosis was defined as CD45+CD66b+Af488+. Neutrophil phagocytosis of A. fumigatus conidia was severely impaired by IBT in a dose-dependent manner when compared with solvent-treated neutrophils (Figure 1E). To rule out stochastic associations of conidia and neutrophils at a superficial level, we used cytochalasin D, an actin polymerization inhibitor, in parallel treatments for each condition tested. In the presence of cytochalasin D, CD45+CD66b+AF488+ events were below 1.35% for solvent-treated neutrophils (Figure 1E), with similar values for all other neutrophil treatments (Supplemental Figure 3). These results indicate that IBT-treated human neutrophils were impaired in their phagocytic capacity as compared with solvent-treated neutrophils.

We next leveraged a neutrophil swarming assay (36) to determine how coordinated chemotaxis to the site of infection and containment of fungal growth may be affected by BTK inhibition. We observed significantly impaired neutrophil swarming over 200 minutes toward A. fumigatus in IBT-treated neutrophils compared with the solvent control (Figure 1, F and G). In addition, we demonstrated that IBT-treated neutrophils were less able to contain fungal growth compared with solvent-treated neutrophils 16 hours after coincubation of A. fumigatus (Figure 1H).

TNF-α compensated IBT-induced defects in neutrophils against A. fumigatus. To better understand how BTK affected the neutrophil immune response against A. fumigatus, we assessed signaling pathways affected by IBT treatments at the transcript level. We collected RNA from unstimulated neutrophils treated with either 0.3 μM IBT or solvent control for 4 hours and assessed the expression of 773 host response genes. Using NanoString nCounter, we detected 18 differentially expressed genes (DEGs) in IBT- versus solvent-treated unstimulated neutrophils (Figure 2A, and Table 1). Interestingly, TNF was the top hit and was downregulated by a log2 fold-change of 4, closely followed by CD274, whose product PD-L1 has been positively correlated with TNF-α production (37, 38). Moreover, RAC2, important for neutrophil granule exocytosis (39) and TNF-α–mediated ROS production (40), was found to be upregulated. Given the role of multiple DEGs in TNF-α signaling pathways, we next examined upregulated and downregulated genes in the TNF-α pathway using a KEGG map (Figure 2B). The analysis revealed that the genes ADGRG3, ALPL, CR1, ERN1, FOS, IL1RAP, IL1RL1, MAP2K4, PIK3CB, RAC2, TIMP2, and TME140 were upregulated or relatively unchanged. Downregulation of APOL6, CD274, FBXO6, GBP1, STAT1, and TNF occurred in IBT-treated neutrophils.

Figure 2

IBT induced downstream upregulation of the TNF-α pathway in human neutrophils. (A) Volcano plot for DEGs in neutrophils treated with 0.3 μM IBT versus DMSO (4.5h, unstimulated). DEGs based on log2 fold change and Padj < 0.05. FDRs were calculated using the Benjamini-Yekutieli method with 3 biological replicates per condition. Red and blue dots represent upregulated and downregulated genes, respectively. (B) TNF-α KEGG pathway was created for all probed genes for IBT-treated neutrophils versus DMSO. Genes in white boxes are genes not included in the nCounter panel. Numbers in circles represent pathways: (1) MAPK signaling pathway; (2) ubiquitin-mediated proteolysis; (3) NF-κB signaling pathway; and (4) PI3K/Akt signaling pathway.

Table 1

DEG from IBT-treated neutrophils versus DMSO-treated neutrophils (unstimulated)

Analysis of transcriptional changes in IBT-treated neutrophils revealed that the TNF signaling pathway was the most affected. We hypothesized that exogenous TNF-α could rescue the immune defects in these neutrophils. Most TNF-α in inflammatory conditions are from heterologous sources (e.g., macrophages, dendritic cells), with a small fraction made from neutrophils. To address their contribution, we quantified soluble TNF-α by ELISA using the supernatant of A. fumigatus–stimulated neutrophils. Indeed, TNF-α levels were 45% lower in IBT-treated cells compared with the solvent control (Supplemental Figure 4A). To test the hypothesis that exogenous TNF-α can restore neutrophil activity against A. fumigatus, we stimulated IBT-treated neutrophils with recombinant TNF-α, and we then challenged them with A. fumigatus. At both 5 ng/mL and 100 ng/mL, TNF-α restored effector activity against A. fumigatus to levels comparable with those of competent neutrophils, as demonstrated by growth inhibition (Figure 3A) and ROS production (Figure 3B and Supplemental Figures 4 and 5). Similarly, TNF-α promoted neutrophil swarming in IBT-treated neutrophils, recapitulating those of control neutrophil treatments (Figure 3, C–E). TNF-α also restored the phagocytic activity of 0.3 μM IBT-treated neutrophils (2.36% phagocytic activity; Figure 1E) compared with 63.6% and 68.2% when 5 ng/mL or 100 ng/mL TNF-α were added, respectively (Figure 3F and Supplemental Figure 5C). We then examined the transcription signature of IBT-treated neutrophils stimulated with A. fumigatus with and without exogenous TNF-α. Out of 773 genes examined by NanoString nCounter, 79 were DEG in IBT-treated versus solvent control-treated neutrophils stimulated with A. fumigatus, 65 of which were compensated (genes not significantly dysregulated for IBT + TNF-α versus solvent control) by 5 ng/mL TNF-α (Figure 3G). Taken together, our data indicate that TNF-α, at doses as low as 5 ng/mL, compensated for IBT-induced defects in neutrophils.

Figure 3

TNF-α rescued IBT-induced immune defects in neutrophils against A. fumigatus. Human neutrophils were treated with 0.03 μM IBT, 0.3 μM IBT, or DMSO for 30 min followed by a 4h incubation with TNF-α and coincubated with A. fumigatus B5233 strain for all figure panels. For all panels, data are representative of at least 3 independent experiments. (A) Neutrophils were incubated with A. fumigatus (MOI:0.25) for 5h, and metabolic activity was measured by resazurin assay. Data calculated through time course study (see raw data in the Supporting Data Values file) and panel represent the output from linear regression analysis using Gompertz fit with percentages of growth inhibition of A. fumigatus by neutrophils in reference to IBT-treated neutrophils. Data are shown as 95% CI, n = 3. Ordinary 1-way ANOVA and Tukey’s multiple-comparison test with a single pooled variance demonstrated a P < 0.001 for all TNF-α treatments versus IBT alone. (B) Neutrophils were stimulated with 1 mg/mL A. fumigatus heat-killed hyphae. ROS production was measured by chemiluminescence using lucigenin. Data are shown as mean ± SD, n = 3. (C) Microscopy panels showing neutrophils swarm formations 200 min after coincubation. (D) Area of neutrophil swarm after 200 min. (E) Area of fungal growth normalized to the growth of A. fumigatus without neutrophils after 16h. Data are shown as mean ± SD, n = 24. Ordinary 1-way ANOVA and Tukey’s multiple-comparison test with a single pooled variance. **P < 0.01; ****P < 0.0001. (F) Neutrophils were coincubated with AF488-labeled A. fumigatus swollen spores (MOI: 10). The displayed percentage of phagocytic neutrophils (CD45-AF700+CD66b-APC+conidia-AF488+) was estimated based on the total number of viable neutrophils (CD45-AF700+CD66b-APC+). At minimum, 10,000 viable CD66b-APC+ events were recorded. (G) Heatmap for DEG based on log2 fold change (1.5 < log2 fold change < –1.5) and a Padj < 0.05. FDR was calculated using the Benjamini-Yekutieli method with 3 biological replicates per condition. RNA from neutrophils coincubated for 5h with A. fumigatus B5233 strain (MOI: 2.5).

In addition to TNF-α, we tested the effect of IFN-γ, G-CSF, IL-1β, and IL-8, on neutrophils treated with 0.3 μM and 3 μM IBT. The effects of GM-CSF on neutrophil function following BTK inhibition is discussed in Desai et al. (41). However, IFN-γ, G-CSF, IL-1β, and IL-8 did not restore neutrophil effector activity but rather further exacerbated the IBT-associated defects for killing capacity against A. fumigatus (Supplemental Figure 6A). Importantly, growing A. fumigatus in the presence of IBT or any of these cytokines alone did not alter the pathogen’s basal metabolic activity (data not shown). While the killing capacity was not compensated by these cytokines, G-CSF mildly improved extracellular ROS production. Similarly, IFN-γ, IL-1β, and IL-8 showed a modest increase (Supplemental Figure 6B). Neither TNF-α nor all other tested cytokines elicited neutrophil ROS production in the absence of a stimulant. Additionally, neutrophil swarming and phagocytosis defects were not improved by exogenous IFN-γ, G-CSF, IL-1β, or IL-8 in IBT-treated neutrophils (Supplemental Figure 6, C–E). These data indicate that TNF-α, specifically, restored the defects caused by BTK inhibition on human neutrophil effector activity.

TNF-α improved effector function defects imposed by other BTK inhibitors. Patients treated with IBT carry an increased risk for invasive fungal infections (15). However, patients on newer agents in this class rarely report significant invasive fungal infections (42–46). It remains unclear whether these agents behave differently with respect to A. fumigatus–specific neutrophil effect activity. To determine if other FDA-approved BTK inhibitors affected antifungal immunity, we used ABT and ZBT, newer-generation BTK inhibitors with reported decreased off-target activity (47, 48). Using the growth-inhibition measurement, both drugs at physiologically relevant concentrations (1 μM for ABT and 0.4 μM for ZBT) (49–52) or 10-fold below disrupted immunological mechanisms implicated in Aspergillus defense (Figure 4A), confirming that BTK inhibition dampened the neutrophil response against A. fumigatus. Therefore, we considered whether TNF-α could compensate the specific defects imposed by ABT and ZBT in neutrophils. We measured A. fumigatus killing, ROS production, phagocytosis, and swarming in ABT- and ZBT-treated neutrophils. These experiments revealed similar out