AKB-4924 pretreatment enhances production of nitric oxide and host defense peptides during UPEC infection.

<p>(<b>A</b>) Nitrite production from AKB-4924 treated 5637 cells was significantly higher than from DMSO treated cells 2 h post-infection. (Uninfected, n = 6; UPEC CFT073 infected, n = 12). Left panel: Absolute nitrite production (μM). Right panel: Relative nitrite productions per log CFU per mL (n = 6). (<b>B</b>) Nitrite released from WT (iNOS +/+) and heterozygous (iNOS +/-) per log CFU per bladder (gram) (n = 4–5) (<b>C</b>) WT (iNOS +/+), iNOS+/- and iNOS-/- C57BL/6 mice were infected with UPEC CFT073 for 18–24h before bladders were harvested for CFU enumeration (n = 7, 6). (<b>D</b>) WT and iNOS +/- C57BL/6 mice were pre-treated with 0.2 mg AKB-4924 or ωehicle for 1 h followed by infection with UPEC CFT073 (n = 4–6). (<b>E</b>) Human AMP β-defensin 2 (hBD2) (n = 8) and cathelicidin LL-37 (n = 7–10) mRNA level in UPEC CFT073 infected 5637 cells. Results were normalized to β-actin. (<b>F</b>) Real-time qPCR shows murine AMP βdefensin 2 (mβD2) and CRAMP mRNA level in bladders (n = 10–12). Error bar = S.E.M **<i>P</i> < 0.01, *<i>P</i> < 0.05, Student’s two-tailed unpaired t-test. (<b>G</b>) Representative images illustrating the distribution of murine cathelicidin (red) and nuclei (DAPI, blue) in the mucosa of UTI89 infected bladders with vehicle or AKB-4924 pre-treatment (n = 6). Upper panel scale bar = 50 μm. Bottom panel: higher magnitude focused on the uroepithelial cell layer of bladder. Scale bar = 25 μm. (<b>H</b>) Representative western blot of mouse bladder left uninfected (n = 2) or infected overnight with UPEC CFT073 (n = 3). Infected animals were pre-treated with vehicle or AKB-4924 for 2 h. Relative level of cathelicidin level was measured using Image J and normalized to β-actin from 2 independent western blot experiments (2–3 different animals in each western blot).