Role of hypoxia inducible factor-1α (HIF-1α) in innate defense against uropathogenic Escherichia coli infection

Uropathogenic E. coli (UPEC) is the primary cause of urinary tract infections (UTI) affecting approximately 150 million people worldwide. Here, we revealed the importance of transcriptional regulator hypoxia-inducible factor-1 α subunit (HIF-1α) in innate defense against UPEC-mediated UTI. The effects of AKB-4924, a HIF-1α stabilizing agent, were studied using human uroepithelial cells (5637) and a murine UTI model. UPEC adherence and invasion were significantly reduced in 5637 cells when HIF-1α protein was allowed to accumulate. Uroepithelial cells treated with AKB-4924 also experienced reduced cell death and exfoliation upon UPEC challenge. In vivo, fewer UPEC were recovered from the urine, bladders and kidneys of mice treated transurethrally with AKB-4924, whereas increased bacteria were recovered from bladders of mice with a HIF-1α deletion. Bladders and kidneys of AKB-4924 treated mice developed less inflammation as evidenced by decreased pro-inflammatory cytokine release and neutrophil activity. AKB-4924 impairs infection in uroepithelial cells and bladders, and could be correlated with enhanced production of nitric oxide and antimicrobial peptides cathelicidin and β-defensin-2. We conclude that HIF-1α transcriptional regulation plays a key role in defense of the urinary tract against UPEC infection, and that pharmacological HIF-1α boosting could be explored further as an adjunctive therapy strategy for serious or recurrent UTI

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

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

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

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