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. 2013 Oct 21;8(10):e77714.
doi: 10.1371/journal.pone.0077714. eCollection 2013.

Expression and antimicrobial function of beta-defensin 1 in the lower urinary tract

Brian Becknell  1 , John David Spencer, Ashley R Carpenter, Xi Chen, Aspinder Singh, Suzanne Ploeger, Jennifer Kline, Patrick Ellsworth, Birong Li, Ehrhardt Proksch, Andrew L Schwaderer, David S Hains, Sheryl S Justice, Kirk M McHugh
Affiliations

Expression and antimicrobial function of beta-defensin 1 in the lower urinary tract

Brian Becknell et al. PLoS One. .

Abstract

Beta defensins (BDs) are cationic peptides with antimicrobial activity that defend epithelial surfaces including the skin, gastrointestinal, and respiratory tracts. However, BD expression and function in the urinary tract are incompletely characterized. The purpose of this study was to describe Beta Defensin-1 (BD-1) expression in the lower urinary tract, regulation by cystitis, and antimicrobial activity toward uropathogenic Escherichia coli (UPEC) in vivo. Human DEFB1 and orthologous mouse Defb1 mRNA are detectable in bladder and ureter homogenates, and human BD-1 protein localizes to the urothelium. To determine the relevance of BD-1 to lower urinary tract defense in vivo, we evaluated clearance of UPEC by Defb1 knockout (Defb1(-/-)) mice. At 6, 18, and 48 hours following transurethral UPEC inoculation, no significant differences were observed in bacterial burden in bladders or kidneys of Defb1(-/-) and wild type C57BL/6 mice. In wild type mice, bladder Defb1 mRNA levels decreased as early as two hours post-infection and reached a nadir by six hours. RT-PCR profiling of BDs identified expression of Defb3 and Defb14 mRNA in murine bladder and ureter, which encode for mBD-3 and mBD-14 protein, respectively. MBD-14 protein expression was observed in bladder urothelium following UPEC infection, and both mBD-3 and mBD-14 displayed dose-dependent bactericidal activity toward UPEC in vitro. Thus, whereas mBD-1 deficiency does not alter bladder UPEC burden in vivo, we have identified mBD-3 and mBD-14 as potential mediators of mucosal immunity in the lower urinary tract.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of BD-1 in the uninfected urinary tract.
(A) Expression of human DEFB1 mRNA (TOP) and mouse Defb1 mRNA (BOTTOM). Samples were normalized for GAPDH / Gapdh content and expressed as fold-difference compared to a pool of uninfected human / mouse bladder cDNA using the 2^-ΔΔCT method[22]. * indicates p < 0.05 in 2-tailed student’s t-tests comparing indicated organ to kidney. The average fold change ± standard error of the mean (S.E.M.) for each organ is shown (n=4 bladders, 2 ureters, 3 kidneys). (B) HBD-1 protein localizes to bladder urothelium by IHC. US: Urinary Space; Uro: Urothelium; M: Muscularis. Similar results were seen in ureter (data not shown). 400x original magnification.
Figure 2
Figure 2. Effect of Defb1 deficiency on UPEC burden.
(A) RT-PCR confirms absent expression of Defb1 mRNA in kidneys of Defb1 -/- mice (-/-, n=2), versus presence of the predict PCR product in wild type kidneys (+/+). Gapdh RT-PCR is included as a loading control. (B) Bacterial burden in bladders and kidneys of wild type (WT) versus Defb1 -/- (knockout, KO) mice at indicated timepoints following UPEC inoculation. Compiled data from at least 2 separate experiments in 8 mice are shown. The horizontal lines indicate geometric means. The dashed horizontal line indicates the lower limit of detection. There was no significant difference between geometric means at any time following infection in bladders or kidneys (Mann-Whitney Test, p > 0.05). (C) Bacterial burden in bladders and kidneys of wild type (WT) versus Defb1-/- (knockout, KO) mice at 24 hpi following inoculation with 5x105 CFU UTI89.
Figure 3
Figure 3. Effect of UPEC infection on Defb1 mRNA expression.
(A) Timecourse of Defb1 mRNA expression in bladders (open boxes) versus kidneys (shaded boxes) of UPEC infected mice. Time in hpi is indicated on the X axis. Samples were normalized for Gapdh mRNA expression and compared to a pool of uninfected bladder or kidney cDNA using the 2^-ΔΔCT method[22]. Means ± S.E.M. are shown (n=4 bladders and 4 kidneys/group) * p < 0.05, student’s t-test, indicated timepoint versus uninfected bladders; # p < 0.05, student’s t-test, indicated timepoint versus uninfected kidneys. (B) Defb1 mRNA was measured in ureters from uninfected C57BL/6 females (shaded box) and 16 hpi with UTI89 (open box). Samples were normalized for Gapdh content and expressed as fold-difference compared to a pool of uninfected mouse ureter cDNA using the 2^-ΔΔCT method. There was no significant difference in Defb1 expression between groups. (C) Bladder Defb1 mRNA expression is down-regulated in C3H/HeN mice 16 hpi, but kidney Defb1 mRNA levels are unchanged. Shaded boxes represent uninfected organs, and open boxes represent organs harvested 16 hpi. Samples were normalized for Gapdh mRNA expression and compared to a pool of uninfected kidney cDNA using the 2^-ΔΔCT method[22]. Means ± S.E.M. are shown (n=3 bladders and 3 kidneys/group) * p < 0.05, student’s t-test, naïve versus infected C3H/HeN bladders.
Figure 4
Figure 4. Additional BDs are expressed in the lower urinary tract.
(A) Expression of Defb mRNAs in uninfected kidneys, ureters, and bladders. Samples were normalized for Gapdh content and compared to a pool of uninfected bladder cDNA using the 2^-ΔΔCT method[22]. Means ± S.E.M. are shown from 4 bladders, ureters, and kidneys. * indicates p < 0.05 in a 2-tailed Student’s t-test comparing the indicated organ to kidney. (B) Defb3 and (C) Defb14 mRNA expression in bladder following UPEC infection. Time in hpi is indicated on the X axis. All samples were normalized for Gapdh content and compared to a pool of uninfected bladder cDNA using the 2^-ΔΔCT method[22]. The horizontal line indicates the geometric mean for each timepoint. Samples on the X-axis had undetectable Defb14 mRNA expression. (n=4 animals/group; Student’s 2-tailed t-test, p > 0.05 for all comparisons of uninfected versus infected timepoints).
Figure 5
Figure 5. Bladder mBD-14 protein expression is induced by UPEC infection.
Naïve and infected bladders harvested at indicated hpi were subject to mBD-14 IHC and counterstained with hematoxylin. Whereas mBD-14 is undetectable in uninfected bladder, it is expressed throughout the urothelium as early as 2 hpi, with increased expression by 6 hpi (black arrows) that persists at subsequent timepoints. 100x magnification.
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