Fig 1.
Vaginal E. coli titers wax and wane in mice not treated with exogenous estrogen.
Seventeen non-pregnant female C57BL/6 Jackson mice were vaginally inoculated with E. coli strain UTI89, and vaginal washes were collected to monitor infection status over 12 days. Each point shows an individual mouse, with lines connecting the titers of a given mouse from one time point to the next. The percentage of mice with any detectable titer is shown in the box at the top of the graph. Data are combined from two independent experiments and zeros are plotted at the limit of detection. These experiments were conducted with static (non-ventilated) micro-isolator cages.
Fig 2.
After exogenous estrogen treatment, some C57BL/6J mice stay in the estrus phase for several weeks.
C57BL/6 mice from Jackson or Envigo were given two doses of β-estradiol 17-valerate 72 hours apart and followed for one month. (A) Vaginal (PBS) washes were collected at the indicated 15 time points over 31 days and the stage of the estrous cycle was determined by wet mount microscopy according to the criteria in [27]. Data are from one experiment with n = 5 mice per group. (B) Shown are wet mount images at 20x magnification, taken at the indicated days post-estrogenization, from one representative Jackson mouse that did not come out of estrus during the experiment (top row) and one representative Envigo mouse that came out of estrus on day 13 (bottom row). Estrus is defined as an abundance of cornified epithelial cells in washes (examples marked with * in the first column); as mice leave estrus these are replaced by more rounded epithelial cells (examples marked with # in the third column) and leukocytes (examples marked with arrows in the third column). These experiments were conducted with static (non-ventilated) micro-isolator cages.
Fig 3.
Clinical uropathogenic E. coli isolates robustly colonize the female reproductive tract of estrogenized mice.
(A) Mouse vaginal E. coli infection model. Non-pregnant female C57BL/6 Jackson mice were given two doses of β-estradiol 17-valerate (E2) and vaginally inoculated with E. coli strains, then followed for 12 days. Vaginal washes were collected to monitor infection status. (B through D) Mice were infected with UTI89. Bacterial titers were enumerated in vaginal and uterine horn tissues after 24 hours (B) and in vaginal washes in 12 day infections (C) and a longer-term (26 day) infection (D). (E through G) Mice were infected with the E. coli strains UTI89 (n = 14), CFT073 (n = 10) or MG1655 (n = 10) for 12 days. Bacterial titers were enumerated in vaginal washes (E), vaginal homogenates (F) and uterine horn (UH) homogenates (G). Data are combined from two or more independent experiments except for (B and D); zeros are plotted at the limit of detection and bars indicate median values. ** P < 0.01, *** P < 0.001, Kruskal-Wallis test with Dunn’s correction. hpi, hours post-infection; dpi, days post-infection. These experiments were conducted with static (non-ventilated) micro-isolator cages.
Fig 4.
Uterine horn titers are significantly positively correlated with titers in the vagina and vaginal washes.
The E. coli titers from all mice shown in Fig 3E–3G were assessed by Spearman correlations comparing vaginal washes vs. vaginal homogenates (A), vaginal homogenates vs. uterine horn homogenates (B), and vaginal washes vs. uterine horn homogenates (C). The first column shows all bacterial strains (UTI89, CFT073, and MG1655) combined, and the second, third and fourth columns show UTI89, CFT073 and MG1655 individually. The Spearman correlation P value is indicated in the top right of each graph. Data are combined from two or more independent experiments and zeros are plotted at the limit of detection. These data come from experiments conducted with Jackson mice housed in static (non-ventilated) micro-isolator cages.
Fig 5.
Ascension to the upper FRT is not due to techniques employed during vaginal inoculation or washing, and E. coli in the FRT can be spontaneously transmitted among estrogenized cage mates.
Shown are bacterial titers in the vagina and uterine horns. (A) Estrogenized mice were vaginally inoculated with 104 CFU of UTI89 and immediately sacrificed. (B) Estrogenized mice were vaginally inoculated with 104 CFU of UTI89 and sacrificed at seven dpi without collecting vaginal washes. (C) In two experiments (the first shown in black and the second in grey), a cage of five mice were estrogenized. Two were mock-infected with PBS and three were infected with 104 CFU of UTI89. At 12 dpi, UTI89 was detected in all infected mice, and was also detected in the vagina of four out of four mock-infected cage mates and in the uterine horns of three out of four mock-infected cage mates. All mice came from the Jackson Laboratory; experiments from panels A and B and replicate one in panel C were performed with static (non-ventilated) microisolator cages, and replicate two in panel C was performed with HEPA-filtered cages with airflow control. Zeros are plotted at the limit of detection and bars indicate median values.
Fig 6.
Vaginal washes from infected mice contain white blood cells.
Vaginal washes from estrogenized Jackson mice were collected at the indicated time points and examined by light microscopy. (A) White blood cells (examples indicated with circles) and exfoliated vaginal epithelial cells (examples indicated with arrows) were observed in washes. Scale bars = 100 μm. (B) Vaginal washes from UTI89-infected and mock-infected (PBS-inoculated) mice were scored for the presence of parabasal cells and white blood cells (WBCs) in a blinded fashion. * P < 0.05, Mann-Whitney U test. These experiments were conducted with static (non-ventilated) micro-isolator cages.
Fig 7.
E. coli infection elicits inflammation in the uterine horns but not the vagina.
Estrogenized Jackson mice were infected with the E. coli strains UTI89 or MG1655, or mock-infected with PBS. After 12 days, inflammation was assessed in vaginal and uterine horn tissues by a blinded assessment of hematoxylin and eosin-stained tissue sections. (A) Representative images of vaginal tissue from mock-infected vs. UTI89-infected mice. Scale bars, 100 μm. (B) Blinded scoring of vaginal inflammation, where 0 = absent (normal tissue with no immune infiltrate in epithelium), 1 = minimal, 2 = moderate, 3 = profound and 4 = severe (profound tissue inflammation plus immune cells crossing the epithelium into the lumen). (C) Representative images of uterine horn tissue from mock-infected vs. UTI89 or MG1655-infected mice. Scale bars on the top row are 100 μm; squares indicate regions shown at higher magnification on the bottom row, with scale bars indicating 20 μm. (D) Blinded scoring of uterine horn inflammation, using the same scoring criteria from panel B. ** P < 0.01, Kruskal-Wallis test with Dunn’s correction. (E and F) Spearman’s correlation was used to compare vaginal wash titers collected at the time of sacrifice to the uterine horn histology scores shown in panel D for mice infected with UTI89 (E) or MG1655 (F). Data are from four independent experiments conducted in both static (non-ventilated) micro-isolator cages and HEPA-filtered cages with airflow control, with similar results between conditions.
Fig 8.
Flow cytometry demonstrates increased neutrophil, monocyte and eosinophil infiltration into the uterine horns upon E. coli infection.
Flow cytometry was performed on vaginal (VG, panel A) and uterine horn (UH, panel B) single cell suspensions from mock-infected (PBS-inoculated) or UTI89-infected Jackson mice. The gating strategy used was: Siglec-F-, Ly6G+ cells were considered neutrophils; Siglec-F-, Ly6G-, CD11b+, CD11c-, Ly6C+ cells were considered monocytes; Siglec-F-, Ly6G-, CD11b+, CD11c-, Ly6C-, F4/80+ cells were considered macrophages; and Siglec-F+ cells were considered eosinophils. * P < 0.05, ** P < 0.01, Mann-Whitney U test. Data are combined from two or more independent experiments; data points represent actual values for each individual mouse and bars indicate median values. These experiments were conducted with static (non-ventilated) micro-isolator cages.