BAY 11-7082

All mice were housed and cared for at the Rangos Research Center of the Children’s Hospital of Pittsburgh (Pittsburgh, PA), and all experiments were approved by the Institutional Review Board of the University of Pittsburgh (Permit number: 12040382). All studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Throughout the course of all experiments, mice were housed with access to food, water and standard bedding. Endotoxemia was induced in all experiments by intraperitoneal injection of LPS (Escherichia coli 0111:B4 purified by gel filtration chromatography, >99% pure, Sigma-Aldrich) at a dose of 3 mg/kg for 6 hours into 6 week old male mice. At the end of each experiment, all animals were euthanized by CO₂ and cervical dislocation. Immediately prior to injection into mice, the compounds were diluted to an experimental concentration of 100 uM in PBS, with the total concentration of DMSO in the final diluted drug at 1%. Compounds were closely examined to insure that no precipitate formed prior to injection and were stored on ice until injection. In all experiments listed, compounds were delivered to 6 week old mice 30 minutes prior to injection with LPS. Control animals not receiving compound received 1% DMSO dissolved in PBS (“vehicle controls”). Where indicated, mice were also injected with LPS along with the NFκB inhibitor Bay-11-7082 (20 mg/kg, 30 min pretreatment i.p., Cayman Chemical). In addition to assessing the effect on clinical activity of the mice in which the degree of piloerection, tachypnea and movement activity (huddled in the corner versus roaming freely) were assessed, LPS and individual compounds were also injected into NFκB-luciferase reporter mice, in which NFκB is upstream of the luciferase gene (strain NFκB-RE-luc, Taconic Farms Inc, Hudson, NY). In these studies, 6h after LPS injection, mice were administered an i.p. injection of luciferin (160 ug/kg, Caliper Life Sciences), then after 10 minutes, a whole animal image to evaluate luciferase activity was obtained using the IVIS Lumina 3D Optical in vivo imaging system (Caliper Life Sciences, Hopkinton, MA) under 1.5% isofluorane anesthesia. Prior to being euthanized, mice from the above experiments were anesthetized with 1.5% isofluorane and a retro-orbital sinus puncture was performed to obtain a blood sample; serum was obtained via centrifugation and ELISA was performed to assess IL-6 expression (R&D Biosystems). The extent of expression of the pro-inflammatory cytokines IL-6 and iNOS within the intestinal mucosa was determined by RT-PCR (see below).

The study protocol involving the use of human blood cells was approved by the Ethics Committee of the University of Naples Federico II. Cells were isolated from buffy coats of healthy donors. Blood was layered onto Histopaque-1077 (Sigma-Aldrich) and mononuclear cells were collected at the interface. Monocytes were further purified with anti-CD14 Microbeads (Miltenyi Biotec). Purity of cell preparations was >95% as assessed by flow cytometry. Cells were cultured in cIMDM-5 [IMDM, 5% FCS, 1× non-essential amino acids, 1× UltraGlutamine, 25 mM HEPES, 5 µg/mL gentamicin (Lonza)] in 96-well flat-bottom plates (10⁵ monocytes/well) in a final volume of 250 µL. For experiments involving flow cytometry, cells were cultured in suspension (1.5 mL tubes) in cIMDM-5 at a concentration not greater than 2 × 10⁶ cells/mL, then spun down and collected for subsequent experiments.
Cells were treated with different combinations of: LPS (Escherichia coli 026:B6) 10 ng/mL (Sigma-Aldrich), IL-3 5 ng/mL (Peprotech), M-CSF 25 ng/mL, GM-CSF 5 ng/mL (Miltenyi Biotec), P3CSK4 10 ng/mL, Poly(I:C) 1 µg/mL, flagellin 10 ng/mL, imiquimod 1 µg/mL, ODN2006 1 µM (Invivogen), BAY11-7082 1 µM, SP600125 2 µM, rapamycin 50 and 250 nM, torin1 10 and 50 nM, AGK2 10 µM, APO866 0.1, 1, and 10 nM, TG101348 125, 250, and 500 nM (Selleckchem), U0126 2 µM, LY294002 10 µM, SB203580 2 µM (Cell Signaling Technology), 10058-F4 40 µM, EX-527 500 nM (Tocris Bioscience), 2-Deoxy-d-Glucose 1 mM, Etomoxir 40 µM, BAY 85-3934 1 µM, CAY-10585 10 µM (Cayman Chemical), nicotinic acid 10 µM (Sigma-Aldrich), Pyridone 6 100 nM (BioVision), Trichostatin A (TSA) 5 nM (Calbiochem).

Polyriboinosinic:polyribocytidylic acid (poly[I·C]) (LMW)/LyoVec and Pam3Cys-Ser-(Lys)₄ (Pam3CSK4) (Invivogen) were selected as agonists to activate specific immune pathways on PCLSs. Poly(I·C) (LMW)/LyoVec is a synthetic double-stranded RNA (dsRNA) polymer that is complexed with a transfection reagent, and it is sensed by cytosolic helicases retinoic acid-inducible gene I and melanoma differentiation-associated gene 5 (RIG-I/MDA-5) in a specific manner (68 (link)) and forms known essential signaling pathways upon influenza virus infection (69 (link)). Experimental infection in calves with IDV also suggests the activation of this pathway (29 (link)). Pam3CSK4 is a synthetic triacylated lipopeptide that is a potent activator of NF-κB pathway upon binding of TLR2/TLR1 receptor and a MyD88-dependent activation, as described during Mycoplasma spp. infection in different species (41 (link)– (link)43 (link)). To inhibit NF-κB pathway, BAY 11-7082 (Invivogen) was used. BAY 11-7082 was described to inhibit the phosphorylation of IκB-α (which is essential for the release of NF-κB from the cytosolic IκB-α/NF-κB complex) (70 (link)), but it was also suggested to inhibit the inflammasome responses indirectly by preventing the nuclear translocation of NF-κB at the priming step but also to directly inhibitory functions on the NLRP3 inflammasome by blocking the sensor’s ATPase activity (71 (link)). For stimulations, the PCLSs were treated at 0 h with poly(I·C) at a final concentration of 500 ng/mL, followed by stimulation with Pam3CSK4 at a final concentration of 100 ng/mL 24 h later. To inhibit the NF-κB pathway, the PCLSs were pretreated with BAY 11-7082 at a final concentration of 100 ng/mL, followed by M. bovis infection 6 h later.

RAW264.7 cells (2 × 10⁵ cells/well) were seeded in 24-well plates. After 12 h, cells were co-transfected with 0.25 μg of Renilla-expressing plasmids (pRL-SV40-C; Beyotime Biotechnology, Shanghai, China) and either 1 μg of NF-κB binding site reporter plasmids (pNF-κB-TA-Luc; Beyotime Biotechnology, Shanghai, China) or 1 μg of TNF-α promoter reporter plasmids (pTNF-α-promoter-Luc; Beyotime Biotechnology, Shanghai, China) using TurboFect (Invitrogen, Carlsbad, USA). At 4 h post-transfection, cells were treated with PBS, 100 ng/mL ET(H351A), 100 ng/mL ET, 10 μM BAY 11-7082 (BAY), and 40 μM H89, in the presence or absence of 10 ng/mL LPS or 10 ng/mL TNF-α. Luciferase activity levels were determined using the Dual-Luciferase reporter assay system (Promega, Madison, WI, USA) using a microplate luminometer (GLOMAX96; Promega, Madison, WI, USA), following the manufacturer’s instructions. Firefly luciferase activity was normalized against Renilla luciferase activity.