C57BL/6J mice were purchased from CLEA Japan (Japan). Btk KO mice were generated by the CRISPR/Cas9 system ( Supplemental Methods ). Mice were given pathogen-free conditions, and all studies and procedures were approved by Kyushu University Animal Experiment Committee. All animal experiments were conducted in accordance with ARRIVE guidelines and the ethical guidelines of Kyushu University.

For flow cytometry, single-cell suspensions prepared from BM, spleen, and PEC were blocked by using FcR-blocking reagent (TONBO) and stained with fluorochrome-conjugated antibodies ( Supplementary Table 2 ) and streptavidin (BioLegend). Dead cells were stained with 1 μg/ml propidium iodide (Nacalai Tesuque). Stained cells were analyzed on a Cytoflex (Beckman Coulter).

Single-cell suspensions were isolated from spleen and PEC. MZ B cells, FO B cells and Mac were sorted using FACSMelody (BD Biosciences). Cells were blocked by FcR-blocking reagent (TONBO) and stained with the following fluorochrome-conjugated antibodies ( Supplementary Table 2 ) and streptavidin (BioLegend). For B cell isolation, splenic B cells were purified by negative selection of CD43⁺ cells with anti-CD43 magnetic beads (Miltenyi Biotec). The enriched B cell population was >95% positive for CD19 staining.

Mice were given i.p. with 100 μg of NP-CGG in alum or 50 μg NP-Ficoll. For secondary immunization, mice were given i.p. with 100 μg NP-CGG without adjuvant. Mice were treated with acalabrutinib diluted in drinking water ( Supplementary Methods ). Levels of NP-specific antibodies were analyzed on 96 well plates coated with NP (29)-BSA (Biosearch Technology) and detected with HRP-conjugated goat anti-mouse IgM, IgG1, IgG3 (Southern Biotech). The amount of IL-6 in the culture supernatant was measured by ELISA according to the manufacturer’s protocol (BioLegend).

Mice were given i.p. injection of PBS or 20 mg/kg LPS (O55:B5, Sigma-Aldrich) dissolved in PBS. Mice were given p.o. vehicle or 10 mg/kg acalabrutinib ( Supplementary Methods ) at 1 h and every 12 h up to 3 days after LPS injection, and the mortality of mice was monitored.

The BM cells were extracted from C57BL/6J mice by flushing femurs and tibias, and 4 x 10⁵ BM cells were cultured for 7 days at 37°C CO₂ incubator using BMDM culture medium ( Supplementary Methods ).

1 x 10⁵ cells of MZ B cells and FO B cells from spleen, splenic Mac, and PEC Mac were stimulated with 10 μg/ml LPS (O55:B5, Sigma-Aldrich) for 6 h or 24 h in 96 well plate at 37°C in the presence of vehicle or 1, 10 μM acalabrutinib or, 1 μM BAY 11-7082 (Selleck Chemicals). CD43^- B cells were labeled with 20 μM Cell Trace Violet (Invitrogen) for 5 min at R.T. The cells were stimulated with anti-IgM F(ab’)₂ (Jackson), anti-CD40 (HM40-3, BD Biosciences), recombinant mouse IL-2 (R&D), IL-4 (R&D), IL-5 (R&D) for 72 h or 96 h in 48 well plates at 37°C in the presence of vehicle or 1, 10 μM acalabrutinib. Acalabrutinib was dissolved in DMSO and diluted with culture medium ( Supplementary Methods ).

1 x 10⁵ cells of BMDM were stimulated with 1 μg/ml LPS (O55:B5, Sigma-Aldrich) and 20 ng/ml recombinant mouse IFN-γ (BioLegend) for 6 h or 24 h in 96 well plates at 37°C in the presence of vehicle or 1, 10 μM acalabrutinib.

Total RNA was isolated and purified using the RNeasy micro kit (Qiagen) from FO B cells, MZ B cells, splenic Mac, PEC Mac, and BMDM. cDNA was generated using the ReverTraAce qPCR RT Master Mix (TOYOBO). Real-time PCR was performed on a LightCycler 96 (Roche) using Thunderbird SYBR qPCR mix (TOYOBO). The primer sets used in this study were listed in Supplementary Table 3 . The data were normalized to an β-actin reference.

Immunoblotting sample preparation was described in Supplemental Methods . Samples were electrophoresed into SDS-polyacrylamide gel and transferred to 0.45 μm PVDF membranes (Merck Millipore) by electrophoresis. Membranes were blotted with primary antibody and HRP-conjugated second antibody ( Supplementary Table 4 ). For protein visualization, Chemi-Lumi One (Nacalai Tesque) or ECL Prime Western Blotting Detection Reagents (Cytiba) were used as substrates and protein levels were detected using a FUSION SOLO S (VILBER).

GraphPad Prism 8 (GraphPad Software) was used for all statistics. Unpaired student’s t test and two-tailed Welch’s t-test were used when only two groups were compared, and one-way ANOVA/Tukey was used for multiple comparisons. p values < 0.05 were considered to represent significant differences.

We first determined the inhibitory effects of acalabrutinib on Btk and its downstream signaling pathways in vitro and in vivo. A previous report showed that acalabrutinib binds covalently to Cys481 in the ATP-binding pocket of Btk at 1 μM concentration (21). Our study revealed that acalabrutinib efficiently blocks Btk autophosphorylation at Y231 in naive murine splenic B cells treated with a lower concentration of 0.1 μM acalabrutinib ( Supplementary Figure 1A ). Acalabrutinib significantly inhibits the phosphorylation of its direct substrate PLC-γ2 without affecting the phosphorylation of its non-substrate Erk ( Supplementary Figure 1A ). Acalabrutinib also suppressed increased expression of B cell activation markers CD69, CD80, and CD86 on FO and MZ B cells upon B cell receptor (BCR) stimulation ( Supplementary Figure 1B ). Furthermore, we investigated the inhibitory effect of acalabrutinib on Btk in vivo when administered orally (PO) via gavage at a single dose (10 mg/kg body weight) for 3 h ( Supplementary Figure 1C ). B cells from mice treated with acalabrutinib showed impaired BCR-induced CD69, CD80, and CD86 surface expression ( Supplementary Figure 1D ) and Ca²⁺ mobilization on FO and MZ B cells ( Supplementary Figure 1E ). These results suggest that acalabrutinib efficiently inhibits Btk activity both in vitro and in vivo.

To investigate the effects of acalabrutinib on LPS-mediated endotoxic shock in vivo, we injected mice with a lethal dose of bacterial LPS (20 mg/kg body weight) before administering either vehicle or acalabrutinib ( Figure 1A ). We found that the acalabrutinib-treated mice were highly resistant to LPS-induced mortality ( Figure 1B ). To investigate how acalabrutinib controlled mortality, we examined its effect on the production of pro-inflammatory cytokines in MZ and FO B cells and macrophages isolated from LPS-injected mice ( Supplementary Figures 2A, B ). It should be noted that the number of splenic B cells and macrophages was not altered after acalabrutinib administration ( Supplementary Figures 2A, C ). We found that il6 and cxcl10 mRNA expression, which significantly increased in the MZ B cells in the spleen of LPS-injected mice, was pronouncedly suppressed by acalabrutinib treatment. In contrast, tnf expression was not inhibited by acalabrutinib. Although expression of il6, cxcl10, and tnf was enhanced in splenic FO B cells, acalabrutinib failed to suppress their expression ( Figure 1C ). Splenic macrophages also showed upregulated il6 and cxcl10 expression in LPS-injected mice. However, il6 expression was lower compared to MZ B cells and acalabrutinib was ineffective in inhibiting il6 and cxcl10 expression ( Figure 1C ). In intraperitoneal macrophages, the administration of acalabrutinib failed to inhibit il6, cxcl10, and tnf expression ( Figure 1D ). Furthermore, acalabrutinib significantly reduced serum IL-6 levels in LPS-injected mice ( Figure 1E ). These findings suggest that acalabrutinib primarily suppresses endotoxic shock by limiting the expression of pro-inflammatory cytokine and chemokine in MZ B cells.

To understand the direct effect of acalabrutinib on macrophages, we first investigated the responses of the bone-marrow-derived macrophages (BMDM) to LPS and IFN-γ, which are known as M1 polarization conditions, in the presence of acalabrutinib ( Figure 2A ). Acalabrutinib failed to inhibit the expression of il6 and nos2, which indicates M1-macrophage-related phenotype, and the secretion of IL-6 ( Figures 2B, C ). Responses of MZ and FO B cells to LPS were examined in vitro in the presence or absence of acalabrutinib ( Figure 2D ). The LPS-stimulated upregulation of il6 and cxcl10 transcript levels in MZ B cells was substantially suppressed by acalabrutinib ( Figure 2E ). Secretion of IL-6 by MZ B cells was attenuated by acalabrutinib as assessed using ELISA ( Figure 2F ). These results suggest that acalabrutinib may target MZ B cells as cytokine-producing cells.

TLR4 stimulation through LPS in myeloid-lineage cells results in the activation of NF-κB, leading to the production of various pro-inflammatory cytokines such as IL-6 (29, 30). We investigated whether Btk regulates LPS-mediated IL-6 expression via NF-κB signaling in MZ B cells. As we showed in Figures 2E, F , acalabrutinib did not completely prevent the IL-6 production by MZ B cells, probably because it partially inhibits Btk. Therefore, to accurately assess Btk-dependent signaling, we generated Btk-knockout (Btk KO) mice ( Supplementary Figures 3A–C ). We confirmed that these mice have the same phenotype of B cell differentiation as previously reported ( Supplementary Figures 3D–F , Supplementary Table 1 ). We found that mRNA expression and secretion of IL-6 was markedly reduced in Btk-deficient MZ B cells (75% reduction at the protein level) ( Figures 3A, B ), while acalabrutinib lowered it by only about half ( Figure 2F ). To evaluate the signaling pathway required for Btk activity, we isolated MZ B cells from Btk-deficient mice and examined LPS-stimulated NF-κB activation. Upon LPS stimulation, nuclear translocation of p65 was significantly observed in wild-type MZ B cells, but this was suppressed in Btk-deficient MZ B cells ( Figure 3C ). In FO B cells, however, nuclear translocation of p65 tended to be slightly increased but was neither statistically significant nor Btk-dependent ( Figure 3D ). Furthermore, the NF-κB inhibitor, BAY 11-7082, suppressed IL-6 production in MZ B cells ( Figure 3E ). These data suggest that the TLR4–Btk–NFκB signaling pathway is crucial for IL-6 production in MZ B cells.

Acalabrutinib potentially increases the risk of impaired humoral immunity since Btk is essential for B cell development (5). To understand the potential adverse effects of acalabrutinib, we examined B cell development and antibody responses in mice that were administered acalabrutinib for an extended period of time through drinking water ( Figure 4A ). We observed a decrease in the bone marrow pre-B cells and spleen T2 cells after 1 week of treatment, but there was no change in the number of FO B cells and MZ B cells ( Figures 4B, C ; Supplementary Figures 4A, B ). After 3 weeks of treatment, the Pro- to Pre-B cell ratio increased in the bone marrow, and the number of FO B and MZ B cells in the spleen decreased ( Figures 4B, C ; Supplementary Figures 4D, E ). These results are similar to those observed in Btk-deficient mice with the only difference being that acalabrutinib did not affect B1 cells in the peritoneal cavity (PEC) ( Figures 4B, C ; Supplementary Figures 4C, F ), which is a significant source of natural IgM antibodies (31).

As it is believed that sepsis should be treated for a short period of time in the acute phase, it is assumed that acalabrutinib is administered for a short period of time. However, Btk inhibition is typically associated with the risk of immunodeficiency. To investigate the impact of acalabrutinib on antibody immune responses in vivo, mice were administrated vehicle or acalabrutinib via drinking water for a week before immunization with NP-Ficoll, which is a T cell-independent antigen ( Figure 5A ). We found that antigen-specific IgM and IgG3 antibody production decreased substantially following acalabrutinib treatment, although this reduction was not observed when acalabrutinib was administered 1 week after immunization ( Figure 5B ). The chronic extended use of acalabrutinib, particularly in individuals with chronic lymphocytic leukemia (CLL), may result in immunodeficiency, which is a risk factor for infections. To investigate the impact of long-term treatment of acalabrutinib, we next examined T cell-dependent (TD) antibody responses after immunization with NP-chicken-γ-globulin (CGG) and monitored the production of antigen-specific IgM and IgG1 antibodies ( Figure 5C ). We found that acalabrutinib did not affect IgM antibody responses regardless of whether acalabrutinib was administered 1 week before or after immunization ( Figures 5C, D ). However, mice treated with acalabrutinib 1 week before immunization showed a significant decrease in IgG1 antibody production ( Figure 5C ). In contrast, IgG1 primary responses were normal in mice treated with acalabrutinib 1 week after immunization, although secondary responses were slightly impaired ( Figure 5D ). Thus, long-term treatment with acalabrutinib may lead to a reduction in humoral immunity, but short-term treatment had no significant effect, suggesting that Btk inhibitors may alleviate endotoxic shock without compromising humoral immunity. It also implies that acalabrutinib treatment may not exacerbate the infection that caused the sepsis.

We next conducted in vitro B cell stimulation assays to study the direct effects of acalabrutinib on B cells. Acalabrutinib inhibited B cell proliferation induced by anti-IgM stimuli but not by anti-CD40 plus cytokines ( Figure 5E ). Class switch recombination was also not affected; however, plasma cell differentiation was partially inhibited by acalabrutinib following anti-CD40 antibody and cytokine stimulation ( Figure 5F ), suggesting that Btk may signal downstream of CD40 or cytokines for plasma cell development. Given that signals from BCR and CD40 in B cells are vital for antibody production, acalabrutinib may limit Btk activity and reduce antibody production.