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. 2016 Oct 31;2:16196.
doi: 10.1038/nmicrobiol.2016.196.

A Mucosal Imprint Left by Prior Escherichia Coli Bladder Infection Sensitizes to Recurrent Disease

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A Mucosal Imprint Left by Prior Escherichia Coli Bladder Infection Sensitizes to Recurrent Disease

Valerie P O'Brien et al. Nat Microbiol. .
Free PMC article

Abstract

Recurrent bacterial infections are a significant burden worldwide, and prior history of infection is often a significant risk factor for developing new infections. For urinary tract infection (UTI), a history of two or more episodes is an independent risk factor for acute infection. However, mechanistic knowledge of UTI pathogenesis has come almost exclusively from studies in naive mice. Here we show that, in mice, an initial Escherichia coli UTI, whether chronic or self-limiting, leaves a long-lasting molecular imprint on the bladder tissue that alters the pathophysiology of subsequent infections, affecting host susceptibility and disease outcome. In bladders of previously infected versus non-infected, antibiotic-treated mice, we found (1) an altered transcriptome and defects in cell maturation, (2) a remodelled epithelium that confers resistance to intracellular bacterial colonization, and (3) changes to cyclooxygenase-2-dependent inflammation. Furthermore, in mice with a history of chronic UTI, cyclooxygenase-2-dependent inflammation allowed a variety of clinical E. coli isolates to circumvent intracellular colonization resistance and cause severe recurrent UTI, which could be prevented by cyclooxygenase-2 inhibition or vaccination. This work provides mechanistic insight into how a history of infection can impact the risk for developing recurrent infection and has implications for the development of therapeutics for recurrent UTI.

Figures

Figure 1
Figure 1. Mice from different genetic backgrounds with a history of chronic infection have long-lasting, enhanced susceptibility to recurrent UTI caused by clinical uropathogens
a, Time course of sensitization experiments in C3H/HeN mice. Open circles indicate urine collection to monitor infection status. b, For this experiment only, the convalescent period in C3H/HeN mice was extended for 6 months after the initiation of sterilizing antibiotic therapy. Shown is the incidence of chronic cystitis 28 d.p.i. after challenge with 108 c.f.u. UTI89. Sen, sensitized (initial chronic infection); Res, resolved of the initial infection. c, C57BL/6J mice were challenged with one 107 c.f.u. dose of UTI89, 4 weeks after sterilizing antibiotic therapy, and the incidence of chronic cystitis was determined. Mice were initially infected with a single dose of PBS (‘adult naive’) or 107 c.f.u. of UTI89 (‘single, res’), or were superinfected with two doses of 107 c.f.u. of UTI89 24 h apart, which resulted in either chronic cystitis (‘super, chronic’) or resolution (‘super, res’) (Supplementary Fig. 1). d,e, Juvenile (8 weeks old; ‘J. naive’) and adult (16 weeks old, ‘A. naive’) C3H/HeN mice were challenged with 107 c.f.u. of the uropathogenic E. coli isolates UTI89, EC958, CFT073 or 83972, or the Klebsiella pneumoniae isolate TOP52. Headings indicate the type of infection from which these strains were isolated (MDR, multi-drug-resistant). All sensitized and resolved mice in this experiment were initially infected with UTI89 before antibiotic therapy. d, Incidence of chronic cystitis at 28 d.p.i. e, Urine bacterial burden at 24 h.p.i indicative of acute cystitis. Data are combined from two independent experiments except for b, which shows results for one experiment. For urine titres, data points represent actual values for each individual mouse, zeros are plotted at the limit of detection, bars indicate median values and Mann–Whitney U test was used. For the incidence of chronic cystitis, Fisher’s exact test was used and the no. of mice per group is shown at the top of each bar. *P<0.05, **P < 0.01, ***P < 0.001.
Figure 2
Figure 2. Prior UPEC infection results in bladder epithelial remodelling that varies according to disease outcome
a, Scanning electron microscopy was used to visualize the luminal surface of the bladder. Representative images from N = 2 replicates with a total of n = 4 mice per group are shown. Scale bars, 25 µm. bd, Cell morphology and differentiation was assessed via immunofluorescence of paraffin-embedded bladder sections from N=3 staining experiments with bladder sections from n = 3 adult naive and resolved and n = 6 sensitized mice; representative images are shown. In b, uroplakin IIIa is in green, Trp63 in red, keratin 5 in white and nuclei in blue. In c, keratin 20 is shown in white, E-cadherin in green, Trp63 in red and nuclei in blue. Scale bars, 50 µm. d,e, RNA-seq was performed on whole bladders from n = 7 sensitized and n = 6 resolved convalescent mice. In d, 837 genes were significantly differentially expressed in sensitized mice relative to resolved mice (Padjusted < 0.05). Colours indicate each gene’s absolute log2(fold change): grey ≤0.58; orange between 0.58 and 1; and red >1. Labelled genes had absolute log2(fold change) > 1 and −log10(Padjusted) > 10. Significance was determined by a Wald test and adjusted for multiple comparisons using the Benjamini–Hochberg false-discovery rate correction. In e, pathway analysis was used to assess the biological processes enriched in the most significantly differentially expressed genes in convalescent sensitized bladders relative to resolved bladders, and significance was determined by a right-tailed Fisher’s exact test, with Padjusted < 0.05 considered significantly enriched pathways. Shown are the top 25 broad meta-pathways assembled from the specific enriched pathways by Ingenuity IPA, ordered by most significant P value.
Figure 3
Figure 3
Bladder remodelling fundamentally alters acute cystitis pathogenesis upon UPEC challenge, conferring resistance to early bladder colonization. a, Representative SEM images of the luminal surface of bladders at 6 or 24 h.p.i. with 108 c.f.u. UTI89. Curved arrows show bacteria; arrowheads show neutrophils; ‘E’ denotes regions of exfoliation. The third row contains magnifications of regions denoted by white boxes in the second row. Scale bars, 25 µm for low-magnification and 5 µm for high-magnification images. N = 2 experiments with n=3–4 bladders per group. be, Mice were challenged with 107 c.f.u. UTI89 and acute outcomes were evaluated. Pyuria in urine sediments at 6 and 24 h.p.i. was assessed with a semi-quantitative scale (b) (PMN, polymorphonuclear neutrophil); total bladder bacterial burdens were determined at 6, 12 and 24 h.p.i. (c); intracellular bacterial communities (IBCs) were enumerated in mice at 6 and 24 h.p.i. after infection with GFP-overexpressing UTI89 (d); and bladder invasion and intracellular replication were determined at 3 h.p.i. using an ex vivo gentamicin proection assay (e). Data are combined from two to three independent experiments. Data points represent actual values for each individual mouse, zeros are plotted at the limit of detection, and bars indicate median values. *P<0.05, **P < 0.01, ***P<0.001, Mann–Whitney U test.
Figure 4
Figure 4. COX-2-dependent inflammation during acute cystitis in sensitized mice allows UPEC to circumvent the early urothelial resistance to colonization
a,b, Expression of Ptgs2 (COX-2) in bladders at 24 h.p.i. with 108 c.f.u. UTI89. In a, immunofluorescence microscopy images are shown for a mouse monoclonal antibody against COX-2. L, lumen; LP, lamina propria; U, urothelium; arrowheads indicate nests of COX-2 positive urothelial cells; dotted line indicates urothelial basement membrane. Scale bars, 100 µm, n=3–5 bladders per mouse from N = 2 experiments. COX-2 staining was not seen in any resolved bladders and was seen in about half of sensitized and adult naive bladders, of which representative images are shown. In b, qRT-PCR results are shown for bladder RNA (normalized to mock-infected adult naive bladders). Whiskers are min to max values. c,d, Ptgs2 expression at 24 h.p.i. is positively correlated with urine bacterial burden (c) and pyuria (d). e,f, Mice were pretreated with the COX-2-specific inhibitor SC-236, or mock treated, 30 min before challenge with 107 c.f.u. UTI89. In e, pyuria was determined in urine sediments collected at 24 h.p.i. PMN, polymorphonuclear neutrophil. In f, bladder bacterial burden was determined at 24 h.p.i.. g,h, After an initial 2 week chronic infection and 4–5 week convalescent period after antibiotics, sensitized mice were vaccinated (Vax) with FimH or mock-vaccinated (Mock), boosted or mock-boosted and then challenged with 107 c.f.u. UTI89 (Supplementary Fig. 14). Urine bacterial burden at 24 h.p.i. (g) and incidence of chronic cystitis at 28 d.p.i. (h) were determined. Data are combined from two to three independent experiments. Data points represent actual values for each individual mouse and bars indicate median values. For urine and bladder titres, zeros are plotted at the limit of detection. The Mann–Whitney U test was used for qRT-PCR and urine and bladder titres. For XY correlations a Spearman correlation was used. For incidence of chronic cystitis, Fisher’s exact test was used and the no. of mice per group is shown at the top of each bar. *P<0.05, **P < 0.01, ***P < 0.001.

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