Pharmaceutical evaluation of tirabrutinib

Tirabrutinib was orally administered to male Sprague–Dawley (SD) rats daily for 4 weeks at 3, 10, 100, and 1000 mg/kg. Methylcellulose solution (0.5%, w/v) was administered to the control group. Blood was drawn from the inferior vena cava under anesthesia. B lymphocytes (CD45RA+ and CD3− cells) were identified by a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and CellQuest version 3.3 (BD Biosciences). For this purpose, fluorescein isothiocyanate (FITC)-conjugated anti-CD45RA antibody (clone OX-33; BD Biosciences) and allophycocyanin-conjugated anti-CD3 antibody (clone 1F4; BD Biosciences) were used.

In other experiments, tirabrutinib was orally administered to SD rats daily for 4 weeks at 0.3, 1, 3, 10, and 30 mg/kg. Methylcellulose solution (0.5%, w/v) was administered to the control group. Rats underwent laparotomy under anesthesia and were sacrificed by exsanguination 24 h after the final dosing. Lymph node cells and peripheral blood mononuclear cells (PBMCs) were treated with phosphatase inhibitor cocktail 2 (Sigma-Aldrich, St. Louis, MO, USA), stimulated by 3 mM hydrogen peroxide (H2O2) for 10 min at 37 °C, and stained using anti-phosphorylated Btk (Btk-pY223) antibodies (Cell Signaling, Danvers, MA, USA). The mean fluorescence intensity (MFI) of Btk-pY223 in B lymphocytes (identified by gating CD45RA+ and CD3− cells) was analyzed by flow cytometry (FCM).

Immobilization of MPO and anti-MPO-ICs on slides with chambers

Human native MPO (4 µg/well; Elastin Products, Owensville, MO, USA) was incubated in eight-well chambers on a slide overnight at 4 °C to immobilize MPO on the slide. Alternatively, human native MPO (40 µg/well) was incubated in two-well chambers on a slide similar to immobilized MPO. After washing with phosphate-buffered saline (PBS) plus 0.05% Tween 20 (PBS-T), the slides with chambers were allowed to react with rabbit anti-human MPO polyclonal antibody (0.5 µg/well for eight-well chambers and 5 µg/well for two-well chambers; Abcam, Cambridge, UK) for 1 h at room temperature (RT). PBS-T-washed slides with chambers were used as MPO and anti-MPO-IC-immobilized slides. As a control, rabbit anti-human MPO polyclonal antibody (5 µg) was immobilized on a slide with two-well chambers.

Neutrophil lysates

Neutrophils isolated from the peripheral blood of healthy volunteers using Polymorphprep (Serumwerk Bernburg AG, Bernburg, Germany) were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), applied to two-well chambers on MPO and anti-MPO-IC-immobilized and control slides (1 × 106/well), and allowed to settle at 37 °C. Three minutes later, neutrophils were harvested and lysed in 10 µl lysis buffer for 5 min on ice. The samples were centrifuged at 10,000 rpm for 15 min at 4 °C, and the supernatants were subjected to western blotting. The time course of the neutrophil harvest was determined by preliminary assessments (data not shown).

Western blotting

Neutrophil lysates heated under reducing conditions were applied to 7.5% sodium dodecyl sulfate–polyacrylamide gels and electrophoresed. After transfer onto a polyvinylidene difluoride membrane, the membrane was soaked in PBS-T solution containing 3% bovine albumin serum to avoid nonspecific antibody binding. Immunoblotting was conducted using anti-Btk antibody (1:1000 dilution; Cell Signaling), anti-phosphorylated Btk antibody (1:1000 dilution; Cell Signaling), anti-Vav antibody (1:3000 dilution; GeneTex, Irvine, CA, USA), and anti-phosphorylated Vav antibody (1:1000 dilution; GeneTex) as primary antibodies. These rabbit polyclonal antibodies were reacted at 4 °C overnight. After washing with PBS-T, the membrane was allowed to react with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:10,000 dilution; Jackson ImmunoResearch, West Grove, PA, USA) as a secondary antibody for 1 h at RT. After washing with PBS-T, antibody binding was detected using a chemiluminescent substrate and ImageQuant LAS4000 (Cytiva, Tokyo, Japan).

Suppression of MPO and anti-MPO-IC-induced NET formation by tirabrutinib

Neutrophils isolated from the peripheral blood of healthy volunteers using Polymorphprep were resuspended in RPMI 1640 supplemented with 10% FBS and reacted with tirabrutinib at concentrations of 0, 2.5, 5.0, 10, 25, 50, and 100 nM for 1 h at 37 °C. Neutrophils were applied to eight-well chambers on MPO and anti-MPO-IC-immobilized and IC-nonimmobilized slides (1 × 105/well) and allowed to settle at 37 °C. Four hours later, the samples were fixed with 4% paraformaldehyde (PFA) for 15 min at RT. After removing the chambers from the slides, the samples were mounted with a 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting solution (Vector Laboratories, Newark, CA, USA).

In other experiments, neutrophils pretreated with tirabrutinib were primed with 5 ng/ml TNF-α for 30 min at 37 °C. Cells were applied to eight-well chambers on MPO and anti-MPO-IC-immobilized and IC-nonimmobilized slides (1 × 105/well) and allowed to settle at 37 °C. Four hours later, cells were collected and subjected to FCM to evaluate cell swelling that precedes NET formation [12]. The time course protocol was determined according to the previous study [13].

Image analysis

Five microphotographs were taken randomly. DAPI-positive area/neutrophil was calculated using ImageJ 1.50i (https://imagej.nih.gov/ij/; National Institutes of Health, Bethesda, MD, USA).

MPO-AAV rats

Wistar Kyoto rats (4 weeks old, male) were immunized with human native MPO to induce experimental MPA according to Little’s protocol [14]. In brief, human native MPO (1600 µg/kg) was injected subcutaneously with Freund’s complete adjuvant on day 0, and 800 ng pertussis toxin was injected intraperitoneally on days 0 and 2. Rats were given drug-free feed (n = 6) or tirabrutinib-containing feed (0.0037% or 0.012%) from day 0 (preadministration; n = 8 per group) or from day 28 (postadministration; n = 8/group). Body weight and food intake were measured weekly, and blood was sampled by tail cut every 2 weeks. Blood samples were heparinized, and the separated plasma was stored at − 20 °C until analysis. Urine was collected on day 40. Qualitative evaluation of hematuria and proteinuria was performed using a dipstick (Siemens Healthineers, Erlangen, Germany). All rats were euthanized on day 42 for histological and serological evaluation.

Pathological findings

Formalin-fixed systemic organs were embedded in paraffin. Sections (3.75 µm thick) of formalin-fixed, paraffin-embedded tissues were subjected to hematoxylin-and-eosin staining. Glomeruli exhibiting NCGN, endocapillary proliferation, and tuft necrosis in the maximal section of the kidney were counted, and the glomerular lesion rate (affected glomeruli/total glomeruli) was calculated. Tubular erythrocyte casts in the renal cortex, which represented glomerular bleeding, were also counted. Microscopic foci of pulmonary hemorrhage with regional alveolar bleeding in the maximal section of the lung were counted under a low-power field of view.

MPO-ANCA titer

MPO-ANCA titer was determined using the human MPO-immobilized enzyme-linked immunosorbent assay (ELISA) plate (Euroimmun, Lübeck, Germany) and HRP-conjugated goat anti-rat IgG antibody (Bethyl Laboratories, Montgomery, TX, USA).

NET-forming neutrophils in the blood

Five rats in the control (drug-free) group, six rats in each preadministration group (low and high doses), three rats in the low-dose postadministration group, and four rats in the high-dose postadministration group were randomly selected and subjected to this assay. Rat polymorphonuclear cells were separated from the whole blood on day 42 using Polymorphprep. Cells were resuspended in RPMI 1640 supplemented with 10% FBS and reacted with Sytox Green, a plasma membrane-impermeable DNA-binding dye (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Sytox Green-positive NET-forming neutrophils per 5 × 104 cells in the neutrophil gate were calculated by FCM, as described previously [15].

Immunofluorescent staining for NETs

Frozen kidney sections were subjected to immunofluorescent staining for NETs. The sections were masked with protein block serum-free solution (Agilent, Santa Clara, CA, USA) and allowed to react with rabbit anti-citrullinated histone H3 (Cit-H3) antibody (10 μg/ml; Abcam) for 60 min at RT. The sections were made to react with Alexa Flour 594-labeled donkey anti-rabbit IgG antibody (4 μg/ml; Abcam) and FITC-conjugated mouse anti-MPO monoclonal antibody (2 μg/ml; LSBio, Seattle, WA, USA) for 60 min at RT in the dark. Autofluorescence was suppressed by Vector TrueVIEW (Vector Laboratories). After mounting with a DAPI-containing mounting solution, the sections were observed under fluorescence microscopy.

Statistical analysis

Dunnett’s and Steel’s tests were applied to parametric and nonparametric comparisons among tirabrutinib-administered and control rats. One-way analysis of variance was applied to parametric comparison among multiple groups in vitro. The Mann–Whitney U test was applied to a nonparametric comparison between the two groups in vitro. p < 0.05 was considered statistically significant.