Flagellin

B cells were isolated from WT mouse spleens as described above and plated in 96-well plates at a density of 5 × 10⁶ cells/ml in RPMI 1640 containing 5% fetal bovine serum, 1% L-glutamine, 200 μg/ml penicillin, 200 U/ml streptomycin (all from Corning). Cells were incubated overnight with one of four concentrations of LPS (1 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml), Pam3CSK4 (0.01 μg/ml, 0.05 μg/ml, 0.1 μg/ml, 0.2 μg/ml), flagellin (0.1 μg/ml, 0.5 μg/ml, 2 μg/ml, 10 μg/ml), MALP-2 (10 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml) (all from Novus Biologicals), recombinant mouse heat-shock protein 60 (HSP60) (0.5 μg/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml) (Abcam), recombinant mouse high-mobility group protein B1 (HMGB1) (0.5 μg/ml, 2 μg/ml, 5 μg/ml, 20 μg/ml) (Biolegend), and low molecular weight hyaluronic acid (HA) (0.1 mg/ml, 0.2 mg/ml, 0.5 mg/ml, 1 mg/ml) (R&D Biosystems) (see Fig. 7). Control cells were maintained in RPMI media without stimulation. Cells were then incubated with 5 μg/ml brefeldin A (Biolegend) for 4 hours at 37°C, washed and stained for flow cytometry as described above. All experiments were run at least twice and included at least two technical replicates.

The following antibodies were used: rabbit anti-HO-1 (1:2000 for western blot, Abcam); rabbit anti-COX-2 (1:1000, Cell Signaling Technology); rabbit polyclonal anti-AKT and anti-pAKT (1:1000, Cell Signaling Technology); rabbit anti-actin (1:5000, Santa Cruz); anti-rabbit-HRP (1:2000, Santa Cruz). Pseudomonas aeruginosa (PA) LPS (Sigma-Aldrich) was prepared in PBS at 100X stock solution and used at a concentration of 100 ng/mL. Flagellin (Imgenex, San Diego CA) was used at a concentration of 100 ng/ml). CFTR inhibitor CFTR_inh172, a kind gift from Dr. Alan Verkman)^{32 (link)}, was freshly prepared in DMSO and used at concentration of 20μM. The PI3K/AKT inhibitor LY94002 (Cell Signaling Technology) was prepared in DMSO and used at a concentration of 20μM. The AKT Activator II, SC79 (Merck Millipore) was dissolved in DMSO and used at the final concentrations indicated. Celecoxib (Sigma-Aldrich, St Louis MO) was dissolved in DMSO (stock solution 20 mM) and used at a final concentration of 25μM. For the in vivo study we used the FDA approved branded Celecoxib (Celebrex) at concentration of 25mg/kg/mouse/day. Ibuprofen (Sigma-Aldrich, St Louis MO) was dissolved in DMSO (stock solution 50 mM) and used at a final concentration of 25μM or 100μM. For the in vivo study we used Walgreens Children’s Ibuprofen Suspension (20mg/ml) at a final concentration of 50 mg/kg/mouse/day.