Glybenclamide

All animal experiments, including procedures, sampling and animal cares, were approved by the ethical committee of Kochi Medical School (Japan), in accordance with guidelines and regulations. Mice used in this study were purchased from SLC, Inc (Japan), and were allowed one week for acclimation to the animal facility (Kochi Medical School) before studies were initiated. Peritonitis was induced by injection of 2 mg of ITO-NPs in 1 mL PBS into the peritoneum cavity of male Balb/c mice (7–12 weeks). 6 h post-inoculation, mice were sacrificed and peritoneal cavities were washed with 1 mL of PBS. Peritoneal lavage fluids were centrifuged and supernatants were kept at −20 °C for ELISA. Repeated peritoneal lavage (5 times) with 1 mL PBS was performed to obtain peritoneal cells. Total peritoneal cells were used for flow cytometer analysis. Blockade of NLRP3 inflammasome was achieved by injecting i.p. 200 μL/mouse of a glybenclamide (Sigma-Aldrich) solution in PBS (Gly) representing a dose of 50 mg/kg, at least 45 min before ITO-NP injection.

BAL-derived cells were cultured with RPMI supplemented with 10% FBS, L-Glutamine (2 mM), penicillin (100U/ml) and streptomycin (100 μg/ml) (Sigma-Aldrich, Rome, Italy) in an atmosphere of 5% CO2 at 37°C. Cells were plated for 1 hour to allow macrophages to attach before removing fluttuant cells. Macrophages purity was checked by means of flow cytometry (F4/80⁺CD169⁺Arginase I⁺) and was around 85%, as shown in Supplementary Figure 1B. To verify the phenotype of lung TAMs, we evaluated the expression of arginase I, well-known marker for M2-like macrophages, and iNOS, well-known marker for M1-like macrophages. As shown in Supplementary Figure 1C, the levels of arginase I in lung tumor F4/80⁺ CD169⁺cells were highly increased compared to macrophages in the lung of naïve mice. In contrast, we were not able to detect iNOS levels (data not shown), implying the majority of M2-like cells in the lung of NMU-exposed mice. Then, cells were treated for 5 hours with LPS (0.1 μg/ml), ATP (0.5 mM, for 30 minutes; Sigma Aldrich, Rome, Italy), Glybenclamide (1 μM, Sigma Aldrich, Rome, Italy), Ac-YVAD-cmk (0.1 μg/ml, Sigma Aldrich, Rome, Italy), monoclonal anti-IFNAR antibody (eBioscience, CA, USA).

For pharmacological rescue experiments, 3 days post fertilization zebrafish larvae were treated with 50 µM pinacidil (Sigma-Aldrich P154), 50 µM glybenclamide (Sigma-Aldrich G0639) in 0.1% DMSO or vehicle control combined with 150 ng/ml MS-222 (Sigma) anesthesia in E3 medium 15–30 min prior to high-speed recordings.

RPMI 1640 medium was obtained from Lonza, FBS from Atlanta Biologicals, glutamine and penicillin-streptomycin from Gibco and recombinant murine GM-CSF from PeproTech. Diphenyleneiodonium, N-acetyl cysteine, Glybenclamide, Poly (dAdT), Red Blood Cell Lysing Buffer, and 2-mercaptoethanol were from Sigma, LPS was from Invivogen, Calcein AM from Invitrogen, GeneJuice from Novagen and the Caspase-1 inhibitor ZYVAD-FMK from APEx Bio and Nigericin was from Thermo Scientific, IFN-I receptor subunit 1 (IFNAR1) blocking antibody (MAR1–5A3) and IgG isotype control antibody (MOPC-21) from BioXCell. S. mansoni genomic DNA was from the Schistosomiasis Resource Center (NIH).

Bovine serum albumin (BSA) (A2153), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (C2920), Cyclosporin A (30024), digitonin (D141), 4-(2-hydroxyethyl)piperazine1-ethanesulfonic acid (HEPES) (H3375), rhodamine 123 (R8004), rotenone (R8875), sucrose (S7903), succinate (S3674), SF-6847/Tyrphostin A9 (T182), Trizma Base (93352), glybenclamide (G0639), psora-4 (P9872), quinidine (Q3625), iberiotoxin (recombinant from Mesobuthus tamulus (I5904)), quinine (145904), pinacidil monohydrate (P154), diazoxide (D9035), cgp-37157 (220005), and superoxide dismutase from bovine erythrocytes (S7571) were purchased from the Sigma-Aldrich Corporation (St. Louis, MO, USA). Ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid (EGTA) (A0878,0025) was obtained from PanReac AppliChem ITW Reagents (Darmstadt, Germany). Tetramethylrhodamine, methyl ester (TMRM), Calcein-AM, BCECF-AM (2’,7’-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester, (B1170)), Fluo-5F-AM (F14222) were obtained from Thermofisher (Waltham, MA, USA). Coomassie (Bradford) Protein Quantitative Assay Kit was obtained from Wuhan Servicebio Technology Co. (Wuhan, Hubei, China). Other chemicals were of analytical grade and were purchased from local suppliers.

Potassium superoxide (KO₂) (Alfa Aesar, 96,5%, Ward Hill, MA, USA). Naringenin (Santa Cruz Biotechnology, Inc., 98%, Dalla, TX, USA). Saline (NaCl 0,9%; Fresenius Kabi Brazil Ltda. Aquiraz, CE, Brazil). L-NAME (Research Biochemicals, Natick, MA, USA), KT5823 (Calbiochem, San Diego, CA, USA), ODQ [1H-(1,2,4)-oxadiazolol-(4,3-a)-quinoxalin-1-one, Tocris Cookson, Baldwin, MO, USA]. Glybenclamide, HTAB (Bromide, hexadecyl trimethyl ammonium), dihydrochloride O-dianisidine, GSH (reduced glutathione), EDTA sodium salt, ferric chloride hexahydrate, TPTZ (2,4,6-tripiridil-s-triazine) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-11 carboxylic) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). DMSO 2% and Tween 80 (Química Moderna, Barueri, SP, Brazil).