Reduced brain dissemination and enhanced bactericidal responses in Siglec-E deficient mice upon sublethal GBS challenge.

<p>Comparison of bacterial counts (expressed in CFU) recovered from the kidney (A) and brain (B) of WT mice and Siglec-E KO mice 48 h after intravenous challenge with 1×10⁸ CFU of WT GBS. (C) Brain bacterial counts were corrected for blood contamination (brain/blood ratio) using a conservative estimate of the mouse cerebral blood volume (2.5 ml per 100 g tissue). mRNA expression of IL-1β (D) and IL-12 (E) in lung and IL-10 in spleen (F) was examined by quantitative real time RT-PCR analysis. Results pooled the data from two independent experiment with final numbers of <i>n</i> = 14 for each group. Each circle denotes 1 mouse (A–F). Siglec-E KO microglia cells showed greater bactericidal ability (G) and produced higher levels of TNF-α (H) after GBS challenge. Statistical analysis was performed by Mann-Whitney test (A–F), two-way ANOVA with Bonferroni posttest (G) and one-way ANOVA with Tukey's multiple comparison test (H). *<i>P</i><0.05.</p

Skewed cytokine responses in Siglec-E KO mice challenged in a GBS intranasal pneumonia model.

<p>Mice were infected intranasally with 5×10⁷ CFU WT GBS and cytokine levels in cell-free BAL fluid or lung homogenates collected 6 h post infection. (A) Bacterial load in lung tissue was calculated by dilution plating for CFU enumeration. (B) Cells from BAL were counted and stained with mAb to F4/80 and Gr-1 to quantitate infiltrating cell populations. IL-1β (C) and IL-6 (D) in the BAL and IL-10 in lung homogenates (E) were examined by ELISA analysis. Data shown are means ± SEM and each circle denotes 1 mouse (n = 8 for WT and n = 7 for mSiglec-E KO mice). The difference between different groups was calculated by Mann-Whitney test.</p

Pretreatment with AKB-4924 impairs UPEC urinary tract colonization in C57BL/6 mice.

<p>(<b>A</b>) Bladders from female C57BL/6 mice treated with 1 h vehicle, or 0.2 mg/mL AKB-4924, were recovered after 18–24 h UPEC CFT073 infection. Real-time qPCR shows increased Hif-1 mRNA in AKB-4924 treated animals. (<b>B</b>) Real-time qPCR and ELISA show increased VEGF mRNA and protein expression in AKB-4924 treated group. Data are generated from two independent repeats (n > 5 per experiment). (<b>C</b>) UPEC recoveries from urine (n = 15), bladder (n = 25) and both kidneys (n = 18) of mice that received 1 h of 4924 pre-treatment were significantly decreased compared to vehicle treated mice. Data is presented as mean +/- SEM, generated from 3–4 independent experiments, **<i>P</i> < 0.01. <i>Ex vivo</i> gentamicin protection assay revealed intracellular bacterial CFU from mice 18 h post-infection (n = 10,12)</p

AKB-4924 stabilizes HIF-1α protein and reduces UPEC-mediated infection of cultured human uroepithelial cells.

<p>(<b>A</b>) Western blot illustrating expression of HIF-1α (120 kDa) in the nuclear fraction of uninfected human uroepithelial cell line 5637 left untreated (UT), or treated with DMSO (negative control), DFO (desferrioxamine mesylate; positive control) or AKB-4924 for 2 h. Nuclear matrix protein P84 was used as loading control. Bar graph shows relative intensity of protein bands from repeated experiments (n = 2). (<b>B</b>) Real-time qPCR showing relative HIF-1α and VEGF mRNA expression in uninfected (UI) or UPEC CFT073 infected (2 h) 5637 cells without treatment, or (<b>C</b>) DMSO (mock treatment and 4924 pre-treated cells followed by UPEC infection (n = 10) (<b>D</b>) Bacterial counts from 2 h DMSO or AKB-4924 treated 5637 cells followed by 2 h infection with UPEC CFT073 to measure total bacteria, or 2 h infection with additional 2 h gentamicin (100 μg/mL) treatment to measure intracellular bacteria (n = 3 per group). Shown as mean +/- S.E.M., *<i>P</i> < 0.05, Student’s unpaired t-test. Results are pooled from three independent experiments.</p

AKB-4924 attenuates urinary tract infection.

<p>(<b>A</b>) Bladders isolated from female C57BL/6 mice infected with UPEC CFT073 for 6 h received localized treatment with 0.2 mg of AKB-4924 via transurethral injection for 16 h. Less bacteria were recovered from infected bladders treated with AKB-4924 compared to vehicle. Real-time qPCR shows elevated level of (<b>B</b>) murine AMP CRAMP and (<b>C</b>) mBD2 mRNA (n >11) in AKB-4924 treated animals. Error bar = S.E.M, **<i>P</i> < 0.01, *<i>P</i> < 0.05, Student’s unpaired two-tailed t-test. Results are compiled from 2 independent experiments.</p

Reduced UPEC-mediated inflammatory damage to bladder epithelium with AKB-4924 pretreatment.

<p>(<b>A, B</b>) ELISA analysis shows pro-inflammatory cytokines IL-6, IL-1β or KC in the bladders or kidneys are reduced in AKB-4924 treated mice 18–24 h post UPEC CFT073 infection. Results are compiled from more than two independent experiments (n > 3 per group). (<b>C</b>) Myeloperoxidase (MPO) assay of bladder supernatants (n = 9–11) indicates significant decrease in MPO activity in AKB-4924 pre-treated bladders 18h post UPEC CFT073 infection; Error bar = S.E.M *<i>P</i> < 0.05, Student’s two-tailed unpaired t-test. (<b>D</b>) Representative immunolocalization images of bladder MPO (red) and nuclei (DAPI, blue) of 18 h UTI89-infected mice with or without 4924 pre-treatment (n = 3–4 per group). Scale bar = 50 μm.</p

AKB-4924 reduces UPEC-mediated cytotoxicity and inflammation in human uroepithelial cells.

<p>5637 Cells were infected with UPEC CFT073 for 2h at MOI ~20. (<b>A</b>) Live/dead cell staining illustrates viable (green) or dead (red) cells. Scale bar = 100 μm. (<b>B</b>) Percentage death in uninfected (UI), DMSO and AKB-4924 pre-treated 5637 cells (n > 7). (<b>C</b>) Total attached cells per field of view. Counts were made from multiple random fields of view (10x objective, n > 7) from independent samples (n = 3). Error bar = S.E.M, ***<i>P</i> < 0.001, **<i>P</i> < 0.01, *<i>P</i> < 0.05 by student’s two-tailed unpaired t-test. (<b>D</b>) Western blots of paxillin, phospho-p38 and phospho-p65 expression of uninfected (UI), UPEC-infected 5637 cells with prior exposure of DMSO or AKB-4924 (n = 3 per group). Data represent one of two independent experiments. (<b>E</b>) ELISA detection of pro-inflammatory cytokine proteins IL-6, IL-1β and IL-8 released by DMSO or AKB-4924 pre-treated 5637 cells 2 h post- infection (n = 4 per group). Results are pooled from 3 independent experiments.</p

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).</p

Mice lacking HIF-1 in their bladder epithelium are more susceptible to UPEC urinary tract colonization.

<p>(<b>A</b>) Level of Hif-1 transcripts in bladders of HIF-1^-/- (<i>Hif1αflox/flox</i>/K14-Cre⁺) mice compared to wild-type mice (n = 3). (<b>B</b>) WT or HIF-1^-/- mice were infected with UPEC CFT073 and bladders were harvested 24 h post-infection for CFU enumeration (n = 16). (<b>C</b>) WT or HIF-1^-/- mice were pre-treated with 1 h of 0.2mg of AKB-4924 and infected with UPEC. Bladders were harvested 18 h post-infection for CFU enumeration (n = 7). (<b>D</b>) Images of representative UPEC infected bladders from littermates (n = 3–4 per group) with or without prior AKB-4924 treatment. Error bar = S.E.M ***<i>P</i> < 0.001, **<i>P</i> < 0.01, *<i>P</i> < 0.05, Student’s two-tailed unpaired t-test. Results are mean values from 2 or more independent experiments.</p

Pharmacokinetic properties of CFZ.

<p>(A) Plasma concentration of CFZ or BKI-1294 in mice dosed with 20 mg/kg compound. CFZ was formulated in either corn oil (black) or MC-Tween (gray); BKI-1294 was formulated in 7% Tween 80, 3% ethanol, and 90% water (white). Data shown are mean ± SEM (n = 3). (B) Unchanged CFZ or BKI-1294 recovered in the feces of mice dosed in (A). Recovery was measured each day for three days. Data shown are mean ± SEM (n = 3).</p