Il 1β

Compounds used in the study are as follows: Acycloguanosine, FUDR, Uridine, SP600125, GNE-3511, GSK-J4, L-glutamic acid, and Ivabradine (Millipore Sigma); Forskolin, LY 294002, 666–15, SQ 22536, KT 5720, tetraethylammonium chloride, cesium chloride, OG-L002, S2101, tetrotdotoxin, and ESI-09 (Tocris); 1,9-dideoxy-Forskolin, ZD 7288 and 8-bromo-cyclic AMP (Cayman Chemicals); nerve growth factor 2.5S (Alomone Labs); Primocin (Invivogen); aphidicolin (AG Scientific); IL-1β (Shenandoah Biotechnology); WAY-150138 was kindly provided by Pfizer, Dr. Jay Brown at the University of Virginia, and Dr. Lynn Enquist at Princeton University. Compound concentrations were used based on previously published IC50s and assessed for neuronal toxicity using the cell body and axon health and degeneration index (Supplementary file 1 Table 1 and 2). All compounds used had an average score ≤1. Untreated controls are quantified as ‘Mock’ treatments for all experiments.

THP-1 human monocytes and EA.hy926 human vascular endothelial cells were purchased from the American Type Culture Collection (Manassas, VA, USA). THP-1 cells were grown in RPMI-1640 (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 100 U mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin. For IL-1β treatment experiments, THP-1 cells were plated on 35-mm tissue culture dishes at 5 × 10⁵ cells/dish and then resuspended in FBS-free medium containing the indicated concentration of IL-1β (Wako). EA.hy926 cells were grown in high-glucose (4500 mg L⁻¹) Dulbecco’s Modified Eagle’s Medium (Wako) supplemented with 10% FBS, 100 U mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin. All cells were cultured at 37 °C in a 5% CO₂/95% air environment and sub-cultured every third or fourth day to prevent loss of morphological characteristics.

MIN6 cells or islets were incubated with a cytokine cocktail (DMEM containing 5 ng/mL IL-1β (Fujifilm Wako Pure Chemical Co.), 10 ng/mL TNF-α (Fujifilm Wako Pure Chemical Co.) and 50 ng/mL IFN-γ (Fujifilm Wako Pure Chemical Co.) for 4 h to induce cytokine production within islets (defined as “CC”). The groups incubated with MCC950 (10 µM) or not treated were defined as “CC + MCC950” and “control”, respectively. After treatments, MIN6 cells or islets were lysed with TRIzol Reagent (Thermo Fisher Scientific) and RNA was extracted from lysates using the PureLink RNA Mini Kit (Thermo Fisher Scientific). RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative real-time PCR for cDNA was performed using the LightCycler96 System (Roche) with TB Green Premix Ex Ta II (Takara Bio Inc., Shiga, Japan) for amplification and detection. The expressions of Il1b and Nlrp3 were normalized to the expression of Rplp0. The primers were as follows: 5′-TCCAGGATGAGGACATGAGCAC-3′ (forward) and 5′-GAACGTCACACACCAGCAGGTTA-3′ (reverse) for Il1b, 5′-AATGCCCTTGGAGACACAGGA-3′ (forward) and 5′-TGAGGTGAGGCTGCAGTTGTCTA-3′ (reverse) for Nlrp3, 5′-GGCAGCATTTATAACCCTGAAGTG-3′ (forward) and 5′-TGTACCCATTGATGATGGAGTGTG-3′ (reverse) for Rplp0.

Recombinant mouse cytokines (M-CSF, IFN-γ, IL-4, IL-1β and IL-10) were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant human cytokines (GM-CSF, M-CSF, IFN-γ, IL-4, IL-1β and IL-10) were purchased from Wako (Osaka, Japan).

Normal human bladder epithelial cells (Kurabo Industries Ltd, Osaka, Japan, #KP-4309) were cultured in medium containing UroLife BM (Kurabo Industries, #LUB-LM0054) using a Urolife Comp kit (Kurabo Industries, #LUC-LL0071) at 37°C with 5% CO₂. Cells were seeded in 12-well plates at a density of 5.0 × 10⁴ cells/well, pretreated with RA (Carbosynth) for 1 h at approximately 70% confluence, and then incubated with 10 ng/mL IL1β (FUJIFILM Wako Pure Chemical, #090–06121) for 4 h. After the supernatant had been collected, the cells were washed with phosphate buffered saline (PBS; FUJIFILM Wako Pure Chemical, #166–23555) and RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany, #74104).