Bay11 7082

Freshly isolated human PMNs (2 × 10⁶ cells/ml) were resuspended in PBS, supplemented with 0.2% BSA and 5 mM glucose, and incubated with dihydrorhodamine-123 (2 μM; Invitrogen) for 30 min at RT. Cells were then incubated with control IgG1 or with soluble or immobilized G97-A mAb (1, 2, 5, 10, and 20 μg/ml) for 30 min at 37°C in the absence or presence of N-formyl–Met–Leu–Phe (fMLF, 1 μM; Sigma-Aldrich), before being placed on ice to stop the reaction. The accumulation of H₂O₂ was measured immediately by flow cytometry on a FACSCalibur machine (BD Biosciences, San Diego, CA, USA), and data were analyzed with FlowJo software version 7.6.1 (Tree Star, Ashland, OR, USA). To measure myeloperoxidase (MPO) activity, cell lysate was collected and analyzed using the Myeloperoxidase Activity Colorimetric Assay Kit (BioVision, Milpitas, CA, USA) according to the manufacturer's recommendations.
For signaling inhibitor treatment, PMNs were pre-incubated with the indicated reagents at 37°C for 30 min: PD98059 (20 μM; Cayman Chemical, Ann Arbor, MI, USA), U0126 (10 μM; Promega, Madison, WI, USA), Bay 11–7082 (5 μM; InvivoGen, San Diego, CA, USA), SP600125 (20 μM; Sigma-Aldrich), SB203580 (20 μM; Cayman Chemical) or N-acetylcysteine (NAC, 10 mM; Sigma-Aldrich) before proceeding with the functional assay.

Cells were co-transfected with pLuc or pNF-κB-Luc (500 ng/mL) and pHMCMV-RLuc (160 ng/mL) using Lipofectamine 2000 (Life Technologies). Following 6-hr incubation, the cells were infected with TRADs. After a total 48-hr incubation, luciferase activities in the cells were determined using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA).
For the evaluation of the effects of NF-κB inhibitors on the NF-κB signaling, cells were treated with BAY11-7082 and MG-132 (Invivogen) 24 hr after transfection with plasmids as described above. A luciferase assay was performed 24 hr after the NF-κB inhibitor treatment.

It is well known that poly(I:C) stimulates innate immunity such as TLR3.³³ Thus, it was used for surrogate viral RNA such as rhinovirus.¹³ We also used CpG oligonucleotides (CpG–ODN), TLR9 ligand replacement of viral DNA. Both nucleic acid compounds were purchased from Novus Biologicals. IL‐13 and IL‐4 were purchased from PeproTech. IL‐33 was purchased from Wako Pure Chemical. IL‐37 and CC16 (Clara cell secretory protein; a marker of bronchial lung epithelial cells) were purchased from ProSpec and R&D Systems, respectively. BAY 11‐7082 (an inhibitor of nuclear factor kappa light chain (NF‐κB) enhancer of activated B cells was purchased from InvivoGen. Ruxolitinib, stattic (an inhibitor of signal transducer and activator of transcription 3³⁴), and LY294002 (a phosphatidylinositol kinase‐3 inhibitor) were purchased from Cayman Chemical. The chemotherapy agent, fludarabine, was purchased from Wako Pure Chemical. The corticosteroid, FP, was purchased from Sigma. A type I interferon (IFNs) neutralizing antibody mixture was purchased from PBL Assay Science. Small interfering RNAs (siRNAs) for TLR3, interferon regulatory factor (IRF) 3, RelA (a NF‐κB subunit), JAK1, STAT6, extracellular signal‐regulated kinase (ERK) 1, ERK2, Stealth RNAi siRNA Negative Control Med GC Duplex #2, and Lipofectamine RNA iMAX Reagent were purchased from Invitrogen.