Bruton's tyrosine kinase inhibition effectively protects against human IgE-mediated anaphylaxis

Abstract

No known therapies can prevent anaphylaxis. Bruton's tyrosine kinase (BTK) is an enzyme thought to be essential for high-affinity IgE receptor (FcεRI) signaling in human cells. We tested the hypothesis that FDA-approved BTK inhibitors (BTKis) would prevent IgE-mediated responses including anaphylaxis. We showed that irreversible BTKis broadly prevented IgE-mediated degranulation and cytokine production in primary human mast cells and blocked allergen-induced contraction of isolated human bronchi. To address their efficacy in vivo, we created and used what we believe to be a novel humanized mouse model of anaphylaxis that does not require marrow ablation or human tissue implantation. After a single intravenous injection of human CD34+ cells, NSG-SGM3 mice supported the population of mature human tissue-resident mast cells and basophils. These mice showed excellent responses during passive systemic anaphylaxis using human IgE to selectively evoke human mast cell and basophil activation, and response severity was controllable by alteration of the amount of allergen used for challenge. Remarkably, pretreatment with just 2 oral doses of the BTKi acalabrutinib completely prevented moderate IgE-mediated anaphylaxis in these mice and also significantly protected against death during severe anaphylaxis. Our data suggest that BTKis may be able to prevent anaphylaxis in humans by inhibiting FcεRI-mediated signaling.

Keywords: Allergy; Immunology; Mast cells; Protein kinases.

Figure 1. BTKis abrogate IgE-mediated mast cell and basophil activation and cytokine production in vitro.

(A) Human SDMCs were passively sensitized with 50 ng/mL human biotinylated IgE overnight, then pretreated with BTKis for 15 minutes and activated for 1 hour with 100 ng/mL streptavidin to cross-link IgE. Percentage of total β-hexosaminidase (β-hex) release was determined via colorimetric assay. n = 4–5 using SDMCs from different donors. (B) SDMCs were treated with BTKis and activated as above, then incubated with fluorescently labeled antibodies against LAMP1 and CD203c before analysis by flow cytometry. Percentage of LAMP1⁺ and mean MFI of CD203c were measured in cKit⁺ cells. n = 4 different donors. (C) SDMCs were treated with BTKis for 15 minutes and then washed before IgE cross-linking as above. Twenty-four hours later, cytokine concentrations as indicated were assayed in supernatants using a fluorescent multiplex assay. n = 3–7 different donors. Dotted lines indicate basal secretion by unstimulated cells. (D) To determine the duration of BTKis’ effects, SDMCs were exposed to 1 μM BTKis for 15 minutes and washed at the indicated time points before activation with IgE and assessment of β-hex release as above. n = 3 different donors. (E) The indicated BTKis were added to anticoagulated human whole-blood samples for 15 minutes before activation with anti-FcεRIα antibody (solid lines) or fMLP (dashed lines) as a control. Basophil activation was assessed by CD63 surface upregulation by flow cytometry. n = 4–6 different donors. All data are displayed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with vehicle-treated cells by 2-way ANOVA with repeated measures.

Figure 2. Ibrutinib effectively blocks anti-IgE–induced contraction of human bronchi.

Isolated human bronchi were pretreated with ibrutinib or vehicle for 30 minutes, and then anti-IgE–induced contraction was measured. (A) Cumulative concentration-response curves for anti-IgE–induced contractions are shown for vehicle- and ibrutinib-treated tissues. Contraction is expressed as a percentage of the maximal obtainable contraction evoked by carbamylcholine (100 μM) added at the end of the experiment. n = 4 using bronchi from different donors. (B) Percentage of maximal contraction as achieved by treatment with exogenous histamine is shown for tissues treated with vehicle or 1 μM ibrutinib. n = 2 from different donors. All data are displayed as means ± SEM. ****P < 0.0001 compared with vehicle-treated tissues by 2-way ANOVA with repeated measures. NS, not significant by 2-tailed paired Student’s t test.

Figure 3. HSC-engrafted NSG-SGM3 mice have detectable circulating human leukocytes as early as 4 weeks after HSC injection.

NSG-SGM3 mice underwent a single i.v. injection of cord blood HSCs. (A) Whole-blood samples taken at 4, 8, 12, and 16 weeks after HSC injection were incubated with fluorescently conjugated antibodies against a panel of human (h) and murine (m) cell surface markers as indicated and analyzed by flow cytometry. Representative flow plots from 3 separate experiments are shown for each time point. Quadrant and gate population percentages represent the percentage of the parent gate. (B–I) Percentages of cells that are human (B–G) and murine (H and I) are displayed as leukocyte subsets at the indicated time points after HSC injection; n = 5–8 mice per group. WT mice (C57BL/6J) and non-engrafted NSG-SGM3 mice are included as controls. All data are displayed as means ± SEM.

Figure 4. HSC-engrafted NSG-SGM3 mice support the accumulation, growth, and maturation of human tissue-resident mast cells.

NSG-SGM3 mice underwent a single i.v. injection of cord blood HSCs. At 16 weeks after HSC injection, mice were sacrificed, and whole blood and organs were harvested and processed for detection of human and mouse mast cells and blood basophils/mast cell precursors. Single-cell suspensions were incubated with the indicated fluorescently conjugated antibodies and analyzed by flow cytometry. Cells were gated on mouse CD45⁺ (mCD45) versus human CD45⁺ (hCD45), and then among human cells, human cKit⁺ (hcKit) and human FcεRI⁺ (hFcεRI) cells were analyzed for CD203c and Siglec-8 expression. Representative flow plots from 3 separate experiments are shown for spleen, bone marrow, peritoneal lavage, and whole blood (A) and for various indicated solid organs (B). Quadrant and gate population percentages represent the percentage of the parent gate. (C) Mast cell precursors were quantified as a percentage of hCD45⁺ cells in blood and bone marrow; n = 3 mice per group. (D and E) Mature human and murine tissue-resident mast cells were quantified as a percentage of hCD45⁺ or mCD45⁺ cells, respectively (D), and of total cells (E); n = 3 mice per group. All data are displayed as means ± SEM.

Figure 5. HSC-engrafted NSG-SGM3 mice serve as a robust model of human anaphylaxis.

(A) Schema of engraftment and PSA protocol. (B) Engrafted NSG-SGM3 mice were sensitized with a single i.v. injection of 1.6 μg of human anti-NP IgE and then 24 hours later were challenged with a single i.v. injection of 5, 20, or 500 μg of NP-BSA to induce mild, moderate, or severe PSA, respectively. Mice sensitized with PBS instead of IgE and NSG-SGM3 mice that were not engrafted with HSCs were used as controls. The PSA response was assessed by (i) decrease in core body temperature and (ii) clinical scoring every 10 minutes for at least 1 hour after challenge. Body temperature measurements were ceased after death; therefore, only the surviving mice at each time point are included in averages. All data are displayed as means ± SEM. n = 3–4 mice per group.

Figure 6. Acalabrutinib pretreatment completely inhibits moderate PSA and partially protects against fatal PSA in humanized mice.

(A) HSC-engrafted humanized mice were sensitized and then pretreated with 2 doses of acalabrutinib (1.5 or 15 mg/kg via gavage) or vehicle control at 16 and 4 hours before challenge with 20 μg NP-BSA to elicit moderate PSA. Core temperature drop from baseline (left) and clinical scores (right) are shown as measures of clinical response during PSA. Body temperature measurements were ceased after death; therefore, only the surviving mice at each time point are included in averages. Data are pooled from 3 independent experiments; n = 6–9 total per group. (B and C) To investigate the duration of acalabrutinib’s protection, engrafted humanized mice were sensitized and pretreated with acalabrutinib as described in A, except that NP-BSA challenge was performed either 2 days (B) or 7 days (C) after the last oral dose of acalabrutinib. Data are pooled from 3 separate experiments; n = 11–17 total per group. (D) To investigate acalabrutinib’s ability to prevent fatal anaphylaxis, engrafted humanized mice were sensitized and pretreated with 15 mg/kg acalabrutinib or vehicle, except a higher challenge dose (500 μg NP-BSA) was given to elicit a more severe PSA response. Data shown are pooled from 6 separate experiments; N = 24–27 total per group. (E) The Kaplan-Meier survival curve from experiments in D is shown for both the acalabrutinib- and vehicle-treated groups. All data were analyzed using 2-way ANOVA with repeated measures with the exception of the mortality rate in E, which was analyzed using χ² analysis. All data are displayed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with PBS control group.