Fig 1.
The C3H/HeN mouse model of chronic and recurrent cystitis using the clinical cystitis isolate UTI89.
(A) In the inbred mouse strain C3H/HeN, inoculation with 108 CFU of the clinical UPEC isolate UTI89 can trigger a cyclooxygenase-2 (COX-2)-dependent acute host-pathogen checkpoint (B) comprising high urine bacterial titers, elevated proinflammatory cytokines in the serum, polymorphonuclear neutrophils (PMNs) in urine, and severe bladder inflammation and mucosal wounding [14,17]. (C) Mice that trigger the checkpoint develop chronic cystitis (persistent bacteriuria ≥104 CFU/ml urine coupled with bladder titers ≥104 CFU and bladder inflammation at sacrifice) lasting for four weeks or more [14]; (D) mice that do not trigger the checkpoint spontaneously resolve the infection and develop sterile urine [14]. (E) In mice that develop chronic cystitis, antibiotic therapy administered at four weeks post infection sterilizes the bladder; however, we found that the bladder does not return to its naive state. Instead, the bladder harbors a “molecular imprint” of infection, comprising changes to the bladder transcriptome [15], smaller superficial cells with altered differentiation [15], a remodeled epithelial membrane proteome [17], and the presence of lymphoid follicles [14]. When “challenged” with a second bacterial exposure (typically 107 CFU), these remodeled bladders respond differently from age-matched naive bladders: if cyclooxygenase-2 (COX-2)-mediated inflammation and checkpoint activation is triggered [15,17] (F), then (G) the host develops severe recurrent chronic cystitis [14], which can be caused by UTI89 or other uropathogens such as the urosepsis isolate CFT073 [15]. During infection in the remodeled bladder, the bacterial niche is altered relative to the naive bladder (intracellular replication is reduced) and the host responds with COX-2-mediated PMN infiltration and exfoliation of epithelial cells [15]. In the present study, we investigated whether infection of juvenile C3H/HeN mice with CFT073 would result in the same constellation of phenotypes seen with UTI89 infection, i.e. checkpoint activation, chronic cystitis and bladder remodeling, and susceptibility to recurrent UTI.
Fig 2.
CFT073 infection is less robust than UTI89 at 28 dpi and clears over time.
Juvenile Naive (7–8 week old) female C3H/HeN mice were infected with 108 CFU UTI89, CFT073, or CFT073ΔfimH::FimHUTI89 (“CFT073-FimH”) and monitored for 28 days. (A) Percentage of mice (infected as indicated) that developed chronic cystitis, which was defined as persistent, high-titer bacteriuria (>104 CFU/ ml urine), bladder bacterial burden >104 CFU/bladder and bladder inflammation at time of sacrifice [14]. * P < 0.05, ** P < 0.01, Fisher’s exact test. (B-D) Bacterial burdens (B) and edema (C) and kidney burdens (D) at 28 dpi in mice with chronic cystitis caused by UTI89 or CFT073 (“Chronic”) compared to mice that had spontaneously resolved these infections (“Resolved”). (E) Scanning electron microscopy (SEM) of bladders harvested from mice with chronic UTI89 or chronic CFT073 cystitis at 28 dpi. Representative images from N = 1 experiment with n = 3 mice per group are shown; scale bars, 50 μm. (F) UTI89 and CFT073 urine titers over time in mice meeting the criteria for chronic cystitis (persistent, high-titer bacteriuria >104 CFU/ ml urine). (G) Mice infected with UTI89 (n = 12), CFT073 (n = 18), or CFT073ΔfimH::FimHUTI89 (n = 15) were followed for 14 weeks. Dashed lines indicate our previously established cutoff for high-titer persistent bacteriuria, 104 CFU/ml urine. Note that 4 out of 18 CFT073-infected mice resolved the infection between weeks 3 and 4 (indicated by the arrow); this “late-resolving” phenotype contributes to the lower incidence of CFT073 chronic cystitis at 4wpi, seen in 2A. (H) SEM at 28 dpi of a mouse that resolved a CFT073 infection between days 21 and 28 (a so-called “late resolver”); scale bar, 50 μm. 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, **** P < 0.0001, Kruskal-Wallis test with Dunn’s multiple test correction.
Fig 3.
An acute host-pathogen checkpoint predicts the outcome of CFT073 bladder infection.
At 24 hpi, urine and serum was collected from mice infected with 108 CFU UTI89 or CFT073, and urine bacterial burden (A), pyuria (B) and serum cytokines (C-F) were assessed. Infection outcomes (chronic cystitis vs. resolution) were determined over the course of four weeks, and data are plotted according to the four week infection outcome. There were no statistically significant differences in the overall data (i.e. all UTI89-infected mice vs. all CFT073-infected mice) at 24 hpi. 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, **** P < 0.0001, Kruskal-Wallis test with Dunn’s multiple test correction. PMN, polymorphonuclear neutrophil.
Fig 4.
Chronic CFT073 cystitis causes bladder remodeling phenotypes that persist after antibiotic therapy.
Mice were initially mock-infected (“Adult Naive”), or infected with 108 CFU CFT073, and followed for four weeks, at which time ten days of antibiotics were initiated. Four weeks after the start of antibiotic therapy, convalescent bladders were harvested and assessed by microscopy. (A and B) Scanning electron microscopy (SEM) was used to assess bladder epithelial (urothelial) superficial cell size. (A) Superficial cells from N = 2 replicates with a total of n = 4 mice per group were measured in ImageJ and the average surface area (pixels2) was calculated. Data points represent the average of all measurements for a given mouse. * P < 0.05, Mann-Whitney U test. (B) Representative images from the analysis in (A). Scale bars, 25 μm. (C and D) Cell morphology and differentiation was assessed via immunofluorescence of paraffin-embedded bladder sections from N = 2 staining experiments with bladder sections from n = 3 Adult Naive mice and N = 5 CFT073HC mice; representative images are shown. In (C) uroplakin IIIa is in green, Trp63 in red, keratin 5 in white and nuclei in blue. Scale bars, 50 μm. In (D), keratin 20 is shown in white, E-cadherin in green, Trp63 in red and nuclei in blue. Scale bars, 100 μm.
Fig 5.
CFT073HC mice are protected against CFT073 rUTI, but not against UTI89 rUTI.
Mice were initially infected with 108 CFU UTI89 or CFT073 and monitored over four weeks, at which time antibiotic therapy was initiated. Those mice that developed chronic cystitis during the initial infection were then challenged four weeks after antibiotics (A-C) or six months after antibiotics (D) with 107 CFU UTI89 or CFT073 as indicated. Initially mock-infected mice were included as a control (“Adult Naive”). (A) Mice were sacrificed six hours after infection with CFT073 and bladder and kidney titers were assessed. * P < 0.05, Kruskal-Wallis test with Dunn’s multiple test correction. (B and C). Mice were challenged with UTI89 or CFT073 and followed over four weeks. (B) Urine titers at 24 hours post challenge, representing acute cystitis. (C) Incidence of chronic cystitis over the four week challenge. * P < 0.05, Fisher’s exact test. (D) Incidence of chronic cystitis over the four week challenge, six months after the initiation of antibiotics, compared to age-matched (“Naive”) mice and mice that had resolved their initial infection (“UTI89HR”). * P < 0.05, Fisher’s exact test. # denotes the number of mice per group. Data are 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.
Fig 6.
Depletion of CD4+ and CD8+ T cell subsets increases chronic and recurrent CFT073 cystitis.
Mice infected with 108 CFU UTI89 or CFT073 were given weekly doses of anti-CD4 and anti-CD8 antibodies, or isotype control, and followed for four weeks. (A) The incidence of chronic cystitis was determined at 28 dpi; # indicates the number of mice per group. (B) Urine bacterial burdens at 28 dpi. (C) CFT073-infected mice from (A) were binned according to the length of their infection. ** P < 0.01, Fisher’s exact test for resolving 8–28 dpi. (D) CFT073 urine titers over the course of infection from isotype-treated (left panel) and anti-CD4/8-treated (right panel) mice. Dashed lines indicate our previously established cutoff for high-titer persistent bacteriuria, 104 CFU/ml urine. (E) Shown are the percentages of mice with persistent high-titer CFT073 bacteriuria (>104 CFU/ml urine) over time. (F-J) At 28 dpi, antibiotic therapy was initiated. Those mice that developed chronic cystitis during the initial infection were then challenged four weeks after antibiotics with 107 CFU of the same strain as their initial infection. Depletion antibodies were not administered during the challenge infection. (F) The incidence of recurrent chronic cystitis was determined at four weeks post challenge; # indicates the number of mice per group. (G) CFT073 urine titers over the course of the challenge infection in CFT073HC mice, from mice initially isotype-treated (left panel) and initially anti-CD4/8-treated (right panel). Some samples were unavailable at three days post challenge. (H) Shown are the percentages of CFT073HC mice with persistent high-titer recurrent CFT073 bacteriuria (>104 CFU/ml urine) during the challenge infection. (I and J) Shown are bladder and kidney bacterial burdens (I) and bladder edema (J) at time of sacrifice. Data are combined from two to four 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, Mann-Whitney U test. rUTI, recurrent urinary tract infection.