Il 1ra

THP-1 cells were purchased from American Type Culture Collection (Rockville, MD) and cultured in RPMI (Welgene, Daegu, Korea) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY) and 1% penicillin-streptomycin (PS; Lonza, Basel, Switzerland) at 37°C in 5% CO₂. THP-1 cells were differentiated into macrophages by treatment for 3 h with 300 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO). 24h later, the cells were stimulated to activate NLRP3 inflammasome.
For NLRP3 inflammasome stimulation, THP-1-differentiated macrophages were primed with 2 μg/mL LPS (Ultra-pure LPS, InvivoGen, San Diego, CA) for 4 h, and then treated with 5 mM ATP (InvivoGen) for 45 min. After the cells were washed with phosphate buffered solution (PBS) three times washing, the cells were cultured in high glucose DMEM (Welgene) with 2% (vol/vol) heat-inactivated FBS (Gibco) and 1% PS (Lonza) at 37°C in 5% CO₂ until further assays.
For treatment, rapamycin (1 to 1000 nM; Sigma-Aldrich), BafA1 (75 nM; Sigma-Aldrich), 3-MA (2.5 mM; Sigma-Aldrich), IL-1ra (1 to 200 ng/mL; Sigma-Aldrich), recombinant human IL-1β (1 to 1000 pg/mL; Sigma-Aldrich), or SB-208350 (1 to 100 μM; Sigma-Aldrich) were added to the cultures simultaneously with LPS priming step and maintained until assays.

Cells (1 × 10⁶ cells per well) were plated into six-well plates and inoculated with 0.1 or 5 MOI ZIKV in RPMI 1640 medium the next day. After 1 h of incubation, cells were treated with Diacerein (D9302; Sigma-Aldrich, St. Louis, MO) or IL-1RA (SRP3327; Sigma-Aldrich). At the indicated time points, the cells were washed with phosphate-buffered saline (PBS) by pelleting for cell lysates and RNA extraction. The cells were lysed in radioimmunoprecipitation (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA). Total cellular RNA was extracted using an RNeasy minikit (Qiagen, Hilden, Germany). To determine cell viability, cells were monitored using trypan blue dye exclusion.

hAF and hNP cells were cultured in 6-well plates and incubated at 37 ℃ with the IL-1 receptor antagonist IL-1Ra (100 ng/mL), the p38 inhibitor SB203580 (10 uM, Sigma Chemical Co., St. Louis, MO, USA), and IκB kinase inhibitor Bay 11-7082 (5 mM, Sigma) at the indicated concentrations for 1 h before combined-hResistin and IL-1β stimulation.

Five days after recovery, 5 μL of IL1-Ra (Sigma, 5 μg/mL) or saline (Baxter, Deerfield, IL) were infused through the left cannula using an injector (33 gauge) with a polyethylene tube (PE-20 from Small Parts, Inc.) connected to a syringe and mounted on an infusion pump (Harvard Apparatus). The IL1-Ra dose was based on the inhibition of IL-1-induced sickness syndrome and adjusted for the weight of the animal [49 (link)]. The following day, a second, equal dose of IL1-Ra was infused in the left cannula, and 100,000 transfected astrocytes were infused through the right cannula.

Male wild-type C57BL/6J (B6), Fcrγ^–/–, Il1r^–/–, C1qa^–/–, C3^–/–, Mbl^–/–, and Nr4a^–/– mice were obtained from The Jackson Laboratory. IL-1RA (0.2 ng/g body weight i.v.; Sigma-Aldrich) was used for IL-1β–IL-1R antagonism. C1INH (0.4 U/g body weight, Berinert, CSL Behring) and LNP023 (Factor B inhibitor, 30 μg/g body weight, Adooq Bioscience) were injected i.v. in recipients 24 hours before and 1 hour after lung transplantation. IL-1β (10 μg/kg, i.v.; Thermo Fisher Scientific) was injected 8 hours prior to LRA injection. Neutrophils were depleted by using anti-Ly6G antibody (12.5 mg/kg body weight; Bio X Cell, clone 1A8). Complement C5 was inhibited by using a C5-blocking antibody (100 μg/mouse, 1 hour before lung transplant; provided by Alexion Pharmaceuticals). Control mice were treated with the same amounts of IgG isotype control antibody (Bio X Cell). All mice were maintained in a specific pathogen–free facility at the Center for Comparative Medicine at Northwestern University and used for the described experiments at the age of 9–14 weeks and between 24 and 28 g of body weight.