Pfizer and Food and Drug Administration (FDA)-approved drug libraries were kindly provided by the Center for Supporting Drug Discovery and Life Science Research, Graduate School of Pharmaceutical Sciences, Osaka University. Lipopolysaccharide (LPS) from Escherichia coli O111:B4 was purchased from Invivogen (San Diego, CA, United States). Plain and fluorescent amorphous SPs (Sicastar; 500, 1,500, and 3,000 nm in diameter) were purchased from MicroMod (Rostock, Germany). Chemi-Lumi One Super and MSU were purchased from Nacalai Tesque (Kyoto, Japan). Gobi Kosa dust (yellow dust) was purchased from the National Institute for Environmental Studies (Ibaraki, Japan). Adenosine triphosphate (ATP) was purchased from Enzo Life Sciences (Farmingdale, NY, United States). DRAQ7 was purchased from Biostatus (Loughborough, United Kingdom). Bosutinib, cytochalasin D (Cyto D), dasatinib, L-leucyl-L-leucine methyl ester (LLoMe), PD-161570, and iFluor-488-conjugated phalloidin were purchased from Cayman Chemical Company (Ann Arbor, Michigan, United States). PD-166285 dihydrochloride was purchased from Tocris Biosciences (Abingdon, United Kingdom). Dasatinib hydrochloride for in vivo experiments was purchased from MedChem Express (Monmouth Junction, NJ, United States). Hoechst 33342, LysoTracker Deep Red, and ProLong Gold antifade Mountant were purchased from Thermo Fisher Scientific (Waltham, MA, United States). DRAQ5 and the enzyme-linked immunosorbent assay (ELISA) kits for mouse IL-1β were purchased from BioLegend (San Diego, CA, United States). Can Get Signal Immunoreaction Enhancer Solution was purchased from Toyobo (Osaka, Japan). Immobilon Forte Western horseradish peroxidase (HRP) substrate was purchased from Merck Millipore (Burlington, MA, United States). Collagenase (crude type) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and DNase I was purchased from Sigma-Aldrich (St. Louis, MO, United States). The ELISA kits for mouse chemokine (C-X-C motif) ligand (CXCL) 1 and mouse and human IL-1α and IL-1β were purchased from R&D Systems (Minneapolis, MN, United States). The cytotoxicity lactate dehydrogenase (LDH) assay kit (WST) was purchased from Dojindo Laboratories (Kumamoto, Japan).

RAW264.7, a mouse macrophage cell line, and THP-1, a human monocytic cell line, were purchased from Riken (Ibaraki, Japan).

Anti-phospho-Src family (D49G4), anti-RAB5A, member RAS oncogene family (RAB5A) (E6N8S), HRP-conjugated anti-mouse IgG, and HRP-conjugated anti-rabbit IgG antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States). Anti-lysosome-associated membrane protein-1 (LAMP-1) (1D4B) and anti-phosphatidylinositol 4,5-bisphosphate (PIP2) (2C11) antibodies were purchased from Abcam (Cambridge, United Kingdom). Anti-actin antibody (C-11) was purchased from Santa Cruz Biotechnology (Dallas, TX, United States). Alexa Fluor-labeled secondary antibodies and HRP-conjugated anti-goat IgG (H + L) antibodies were purchased from Thermo Fisher Scientific. The Alexa Fluor 488-conjugated anti-mouse CD11b (M1/70), Pacific Blue-conjugated anti-mouse CD45 (30-F11), allophycocyanin-conjugated anti-mouse CD68 (FA-11), fluorescein isothiocyanate-conjugated anti-mouse CD80 (16-10A1), phycoerythrin-cyanine7-conjugated anti-mouse CD86 (GL-1), Alexa Fluor 647-conjugated anti-mouse Ly-6G (1A8), phycoerythrin-cyanine7-conjugated anti-mouse Ly-6C (HK1.4), and phycoerythrin-conjugated anti-mouse major histocompatibility complex class II (M5/114.15.2) antibodies were purchased from BioLegend.

MSU crystals were prepared using a previously described method (Ikoma et al., 2022).

C57BL/6J mice (5-week-old females) were purchased from Japan SLC, Inc. (Shizuoka, Japan). During the experimental period, all mice were housed in standard cages in a temperature-controlled room under a 12-h light/dark cycle at the animal care facility of the Graduate School of Pharmaceutical Sciences, Osaka University. Mice were provided ad libitum access to standard laboratory mouse chow and drinking water.

RAW264.7 cells were seeded at a density of 3.5 × 10⁴ cells/well into glass-bottom 96-well plates and primed with LPS (100 ng/mL) for 16 h in Eagle’s minimal essential medium supplemented with 10% fetal calf serum (FCS), penicillin (100 U/mL), streptomycin (100 μg/mL), and non-essential amino acids (Nacalai Tesque, 100x). Cells were pretreated with each compound (5 µM) from the Pfizer- and FDA-approved drug libraries for 30 min and then stimulated with SPs (500 nm in diameter, 500 μg/mL) in the presence of DRAQ7 (2 µM) and Hoechst 33342 (1 μg/mL) for 2 h. After the supernatants were collected, the cells were fixed with 4% paraformaldehyde for 15 min and then rinsed with ice-cold phosphate buffered saline. Images were acquired from four fields per well using the Cell Voyager CV8000 High Content Screening System (Yokogawa Electric Corp., Tokyo, Japan). Hoechst 33342-positive and DRAQ7-negative cells and Hoechst 33342 and DRAQ7 double-positive cells were considered viable and dead cells, respectively. The cell death rate was calculated by dividing the number of dead cells by the total number of cells using CellPathfinder software (Yokogawa Electric Corp.). IL-1α levels in the cell culture supernatants were measured using ELISA, as described below. A test compound was defined as a hit if the mean percentage inhibition of cell death and IL-1α release was >50%.

To prepare bone marrow-derived macrophages (BMDMs), mouse bone marrow cells were cultured with macrophage colony-stimulating factor (10 ng/mL) in Roswell Park Memorial Institute (RPMI) 1,640 supplemented with 10% FCS, penicillin, and streptomycin. 5 days after culture initiation, cells were collected and used as BMDMs. The BMDMs were seeded at a density of 4 × 10⁵ cells/well in 48-well plates and primed with LPS (200 ng/mL) in RPMI1640 supplemented with 10% FCS for 6 h. The primed cells were pretreated with bosutinib (20 µM), Cyto D (20 µM), dasatinib (20 µM), PD-161570 (20 µM), or PD-166285 (20 µM) for 30 min and then stimulated with SPs (500, 1,500 or 3,000 nm in diameter, 300 μg/mL), MSU (300 μg/mL), yellow dust (500 μg/mL), or ATP (3 mM) for 2 h or with LLoMe (0.5 mM) for 3 h.

THP-1 cells were seeded at a density of 4 × 10⁵ cells/well in 48-well plates and induced for macrophage differentiation by culturing them with phorbol 12-myristate 13-acetate (10 ng/mL) in RPMI1640 supplemented with 10% FCS for 1 day, followed by an additional 2 days of culture in phorbol 12-myristate 13-acetate-free medium. THP-1 cells were primed with LPS (50 ng/mL) in RPMI1640 supplemented with 10% FCS for 16 h. The primed cells were pretreated with dasatinib (20 µM) for 30 min and then stimulated with SPs (1,500 nm in diameter, 500 μg/mL) for 4 h.

The supernatants from each culture condition were collected after centrifuging the samples at 440 ×g and 4°C for 5 min. The supernatants and cell samples were analyzed.

Cell viability was determined by measuring LDH activity in the culture supernatants using the cytotoxicity LDH assay kit (WST) according to the manufacturer’s instructions.

Cell samples were washed and lysed with 30 µL of the lysis buffer containing 62.5 mM Tris–HCl (pH 6.8 at 25°C), 2% (w/v) sodium dodecyl sulfate, 10% (v/v) glycerol, 0.01% (w/v) bromophenol blue, and 42 mM dithiothreitol. Samples were then processed for immunoblotting using a previously described method (Matsui et al., 2022). Each protein was probed with the appropriate antibodies listed in the subsection “Antibodies,” and the blots were visualized using Immobilon Forte Western HRP Substrate (Merck Millipore) or Chemi-Lumi One Super (Nacalai Tesque). Immunoreactive bands were detected using FUSION Solo S (Vilber Lourmat, Collégien, France) and quantified using the ImageJ software bundled with 64-bit Java 8. (Version 1.53t) (National Institutes of Health, MD, United States).

BMDMs were cultured on coverslips and primed with LPS (200 ng/mL) for 6 h. Further, they were pretreated with Cyto D (20 µM) or dasatinib (20 µM) for 30 min and stimulated with fluorescent SPs (1,500 nm in diameter, 20 μg/mL) for 2 h. The stimulated cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with digitonin (50 μg/mL) for 15 min. The membrane-permeabilized cells were stained with iFluor-488-conjugate phalloidin (1000-fold dilution) and DRAQ5 (200 µM) for 30 min. Images were obtained using the Cell Voyager CV8000 High Content Screening system, and the number of phagocytosed SPs per cell was counted using the CellPathfinder software.

BMDMs were cultured on coverslips and fixed with 3% paraformaldehyde. Immunocytochemistry was performed as described previously (Ikoma et al., 2022). Samples were visualized under an inverted fluorescence microscope (DMI6000B; Leica Microsystems, Wetzlar, Germany) and photographed using the Application Suite X software imaging system (Leica Microsystems).

Mice were randomly divided into four groups and anesthetized via isoflurane inhalation. The groups received different treatments as follows: 1) intratracheal administration of phosphate buffered saline; 2) intratracheal SP administration (100 mg/kg); 3) intratracheal SP administration (100 mg/kg) in combination with intragastric dasatinib administration (30 mg/kg); and 4) intratracheal administration of SP (100 mg/kg) in combination with dasatinib (10 mg/kg). Bronchoalveolar lavage (BAL) fluid was collected 12 h after the intratracheal administration of SPs. Subsequently, 1 mL of phosphate buffered saline was flushed into the lungs, with a recovery of approximately 700 µL of BAL fluid. The remaining lung tissues were used for isolating lung leukocytes as described below. In separate experiments, the lungs were collected 12 h after the intratracheal administration of SPs for histological analysis.

Lung tissues were minced using a pair of scissors and subsequently digested with collagenase (2 mg/mL) and DNase I (100 μg/mL) in RPMI1640 medium supplemented with 10% FCS with continuous stirring at 37°C for 60 min. The suspended cells were centrifuged at a density-gradient of 40%–80% (v/v) Percoll. Cells (lung leukocytes) were collected from the interface, washed, and then used in further experiments.

BMDMs were stained with LysoTracker Deep Red according to the manufacturer’s instructions. Lung leukocytes were stained with antibodies against mouse CD11b, CD45, CD68, CD80, CD86, Ly-6C, Ly-6G, and major histocompatibility complex class II, according to the manufacturer’s instructions. CD45⁺ CD11b⁺ Ly-6C^med Ly-6G^high cells were identified as neutrophils (Ikoma et al., 2022). CD45⁺ CD80⁺ CD86⁺ major histocompatibility complex class II^high CD68⁺ cells were identified as M1 macrophages (Supplementary Figure S1) (Haloul et al., 2019; Nascimento Da Conceicao et al., 2021). The data were acquired using a flow cytometer (CytoFLEX; Beckman Coulter, Brea, CA, United States) and analyzed using the FlowJo software (TreeStar, Ashland, OR, United States).

The mouse lungs were fixed for 24 h with 10% formalin and embedded in paraffin. Five-micrometer sections of the mouse lungs were stained with hematoxylin and eosin and observed under the BZ-X800 automated high-resolution microscope (Keyence, Osaka, Japan) with an analysis application.

Mouse CXCL1, mouse and human IL-1α and IL-1β levels in the culture supernatant and BAL fluid were measured using ELISA kits, according to the manufacturer’s instructions.

All data was calculated using GraphPad Prism 8.0 software (Boston, MA, United States). One-way analysis of variance and the Tukey–Kramer post hoc test were performed for multiple group comparisons. Statistical significance was set at a p-value of <0.05.

RAW264.7 cells are a macrophage cell line with impaired apoptosis-associated speck-like protein containing a caspase recruitment domain expression and negligible effects on the NLRP3 inflammasome (Pelegrin et al., 2008). These cells were used to identify inhibitors against particulate-induced NLRP3 inflammasome-independent pyroptosis. Candidate inhibitors were screened by elucidating whether the treatment of LPS-primed RAW264.7 cells with the test compounds before SP stimulation suppressed cell death and IL-1α release. SP-induced cell death was determined using CellVoyager CV8000, which allows high-throughput imaging-based screening. Two nuclear staining dyes, Hoechst 33342 and DRAQ7, were used to determine cell viability. Hoechst 33342 was used to stain the cell nuclei, whereas DRAQ7, a membrane-impermeable dye, was used to stain the nuclei of dead cells. Accordingly, Hoechst 33342-positive and DRAQ7-negative cells and Hoechst 33342 and DRAQ7 double-positive cells were considered viable and dead cells, respectively (Supplementary Figure S2A). Simultaneously, IL-1α levels in the culture supernatants were also measured. Screening of 1,240 compounds present in the Pfizer- and FDA-approved libraries identified two anticancer drugs, bosutinib and dasatinib, which suppressed SP-induced cell death and IL-1α release (Table 1; Supplementary Figures S2B, C). Similar results were obtained for PD-161570, PD-166285, PD-173952, and PD-407824. Interestingly, all the drug candidates targeted Src family kinases (SFKs), well-known signaling factors involved in various cellular functions, including the induction of inflammatory responses, cell proliferation, cell differentiation, and metabolism (Parsons and Parsons, 2004; Byeon et al., 2012).

Further, mouse primary macrophages were used to validate the effects of bosutinib, dasatinib, PD-161570, and PD-166285 (PD-173952 and PD-407824 were excluded because they are not approved yet and exhibit relatively weak suppressive effects against SP-induced pyroptosis). To assess whether these drugs inhibit SP-induced macrophage pyroptosis, we measured IL-1β and LDH release as indicators of pyroptosis, in addition to IL-1α release. LDH is released when the plasma membrane ruptures and is often used to measure the incidence of lytic cell death, including pyroptosis (Kayagaki et al., 2021). All the drug candidates significantly suppressed SP-induced cell death accompanied by IL-1α and IL-1β release in BMDMs (Figures 1A–C). Since dasatinib is clinically well-studied (Araujo and Logothetis, 2010), it was used as a representative of these candidates in subsequent experiments. We investigated whether dasatinib effectively suppresses SP-induced pyroptosis in human cells. Dasatinib was found to successfully suppress SP-induced cell death accompanied by IL-1α and IL-1β release in phorbol 12-myristate 13-acetate-treated macrophage-like THP-1 cells (Figures 1D–F). Furthermore, dasatinib did not affect the viability of BMDMs during the 12 h treatment at concentrations required to inhibit SP-induced pyroptosis (Figure 1G). Therefore, dasatinib and other SFK inhibitors effectively suppressed particulate-induced pyroptosis.

Macrophages were initially primed with LPS to induce intracellular IL-1α and IL-1β expression by activating toll-like receptor 4. However, toll-like receptor 4 increases the levels of active SFKs by enhancing their transcription and phosphorylation at Tyr416 (Boggon and Eck, 2004; Maa and Leu, 2016). Thus, we determined whether SFKs mediate particulate-induced cell death without priming. As a result, dasatinib significantly suppressed SP-induced cell death in BMDMs even without LPS priming, although the unprimed cells did not release IL-1α and IL-1β upon SP stimulation (Figures 2A–C). Immunoblotting revealed the phosphorylation of SFKs at Tyr416 (phosphorylated SFKs; p-SFKs) in unprimed BMDMs (Figures 2D, E). Furthermore, LPS priming significantly enhanced SP-induced cell death of BMDMs and increased p-SFK levels. Interestingly, dasatinib decreased SP-induced cell death rates and p-SFK levels in LPS-primed BMDMs to levels comparable to those observed in dasatinib-treated unprimed BMDMs. SP stimulation tended to slightly decrease p-SFKs levels in unprimed BMDMs, but did not affect those in LPS-primed cells. To summarize, these results suggest that basal p-SFK levels can mediate particulate-induced cell death, and an increase in p-SFK levels via priming is associated with particulate-induced cell death.

Particulates exhibit size-dependent variations in inflammatory properties and cytotoxicities (Kusaka et al., 2014; Nishijima et al., 2017; Chen et al., 2022). We evaluated the suppressive effects of dasatinib on pyroptosis induced by SPs of different sizes. An actin-polymerization inhibitor, Cyto D, almost completely suppressed cell death accompanied by IL-1α and IL-1β release in BMDMs induced by SPs with diameters of 500, 1,500, and 3,000 nm (Figures 3A–C), indicating these SPs were incorporated via phagocytosis. Thus, dasatinib significantly suppressed the cell death of BMDMs accompanied by IL-1α and IL-1β release induced by SPs, the efficacy of which varied with SP size.

Furthermore, dasatinib suppressed cell death and IL-1α and IL-1β release induced by yellow dust, which contains crystalline silica, and MSU crystals (Figures 3D–F), indicating that it suppresses pyroptosis induced by particulates of various materials.

In addition, we determined whether dasatinib suppresses pyroptosis induced by external stimuli other than particulates. ATP activates the NLRP3 inflammasome by inducing mitochondrial dysfunction, resulting in pyroptosis (Yang et al., 2019). However, dasatinib did not suppress ATP-induced cell death and IL-1α and IL-1β release (Figures 3D–F). Therefore, the suppressive effects of dasatinib on pyroptosis are selective and are strongly exhibited during particulate-induced pyroptosis.

We further attempted to elucidate the mechanism underlying the effect of dasatinib on particulate-induced cellular responses. SFKs regulate Fcγ receptor-mediated phagocytosis (Suzuki et al., 2000). Therefore, we investigated whether dasatinib inhibits the phagocytosis of SPs by BMDMs. BMDMs were visualized by staining the nuclei and filamentous actin with fluorescent dyes after stimulation with fluorescent SPs and counting the number of SPs incorporated into the cytoplasm under a microscope (Figure 4A). Cyto D markedly decreased the number of incorporated SPs; whereas, dasatinib had no effects on SP phagocytosis (Figure 4B). Therefore, the suppression of particulate-induced pyroptosis by dasatinib is unlikely to be caused by the inhibition of phagocytic activity against particulates.

Flow cytometric analysis of BMDMs stained with LysoTracker Deep Red, an acidotropic fluorescent dye which labels acidic organelles, including phagolysosomes, was performed to elucidate whether dasatinib suppresses SP-induced phagolysosomal dysfunction (Ikoma et al., 2022). The stimulation of BMDMs with SPs decreased the fluorescence intensity of LysoTracker Deep Red, indicating loss of the interior acidity of the phagolysosomes owing to leakage of their contents (Figures 4C, D). Dasatinib significantly decreased the population of LysoTracker Deep Red-negative cells among SP-stimulated cells. Thus, dasatinib was found to suppress particulate-induced phagolysosomal dysfunction.

Furthermore, we elucidated whether dasatinib reduced lysosomal dysfunction and subsequent pyroptosis induced by the lysosomotropic compound LLoMe. LLoMe accumulates in the lysosomes and is processed by the lysosomal thiol protease dipeptidyl peptidase I (Uchimoto et al., 1999). This process results in lysosomal dysfunction. Dasatinib decreased the population of LysoTracker Deep Red-negative cells among LLoMe-stimulated cells (Figures 4E, F). Furthermore, it significantly suppressed LLoMe-induced cell death and IL-1α and IL-1β release (Supplementary Figures S3A–C). Therefore, these results indicating that dasatinib suppressed pyroptosis by preventing phagolysosomal and lysosomal dysfunction.

Based on the findings of the present study, SFKs potentially mediate the dysfunction of SP-containing phagolysosomes. SFKs, including Src, Fyn, Lyn, and Yes, are recruited to actin-rich phagocytic cups during Fcγ receptor-mediated phagocytosis (Majeed et al., 2001). Therefore, we performed immunofluorescence analysis of BMDMs to investigate the spatio-temporal activity of SFK after the cells phagocytose particulates. In the initial stages of phagocytosis, PIP2 accumulates and initiates actin polymerization to extend the pseudopod and engulf the target (e.g., particulates) (Scott et al., 2005). Without SP stimulation, both PIP2 and p-SFKs were distributed throughout the cytoplasm of LPS-primed BMDMs (Figure 5). SPs were surrounded by PIP2 in BMDMs 15 min after SP stimulation. Furthermore, p-SFK accumulated around some PIP2-surrounded SPs. Dasatinib abolished p-SFK signals but did not affect the intensity or distribution of the PIP2 signals. These observations suggest that p-SFKs accumulate around the phagosomes engulfing particulates during the initial stages of phagocytosis.

Next, we stained p-SFKs with an early phagosome marker, RAB5A, 30 min after SP stimulation. We observed RAB5A-positive vesicles in the cytoplasm of BMDMs without SP stimulation (Figure 6). Most of the engulfed SPs were surrounded by RAB5A, and some of the RAB5A-surrounded SPs were also surrounded by p-SFK. Dasatinib did not influence the intensity or distribution of RAB5A signals. Furthermore, we performed immunostaining for LAMP-1, a lysosomal membrane protein used as a marker for late phagosomes and phagolysosomes (Kinchen and Ravichandran, 2008). We detected LAMP-1-positive vesicles in the cytoplasm of BMDMs without SP stimulation (Figure 7). Thirty minutes after stimulation, SPs were surrounded by LAMP-1, indicating that SP-engulfed phagosomes matured on fusing with lysosomes. However, LAMP-1 was not detected around p-SFK-surrounded SPs. Dasatinib did not affect LAMP-1 distribution after SP stimulation. In summary, p-SFKs may act on particulate-engulfed phagosomes mainly during the early stages of maturation.

Finally, we evaluated the anti-inflammatory effects of dasatinib on SP-induced lung inflammation in mice. In this model, intratracheally administered SPs induce IL-1α release from pulmonary macrophages in an NLRP3-independent manner, resulting in neutrophil recruitment and inflammation (Ikoma et al., 2022). Dasatinib is an orally administered drug. We also tested topical treatment of the lung with dasatinib by its intratracheal administration. Both, intragastric and intratracheal administrations of dasatinib suppressed SP-induced lung inflammation. Dasatinib administrations via these routes significantly decreased IL-1α and IL-1β levels in the BAL fluid of mice after the intratracheal administration of SPs (Figures 8A, B). Furthermore, dasatinib administration through both the routes suppressed the levels of a neutrophil chemoattractant, CXCL1, and decreased the number of neutrophils in the mouse lungs after SP administration, although the inhibitory effect was stronger with intratracheal administration than that with intragastric administration (Figures 8C–E). These routes also suppressed the increase in pro-inflammatory M1 macrophages in the mouse lungs after SP administration (Figures 8F, G). Consistent with these findings, histological analysis revealed that both dasatinib treatment routes suppressed immune cell infiltration into the lungs after SP administration (Figure 8H). In summary, dasatinib effectively suppressed particulate-induced NLRP3-independent lung inflammation.