Fig 1.
Dynamics of Citrobacter rodentium infection in SPF and germ-free wild-type mice.
(A) Schematic of possible routes of epithelial infection shaping A/E lesion development and renewal in the face of continuous epithelial regeneration. Black arrows indicate direction of epithelial cell migration and luminal exfoliation. Green arrows indicate possible routes of bacterial infection, with or without luminal planktonic stage. (B-D) Luminal colonization quantitated by bacterial plating from feces of SPF mice (panel B; n = 12–21 per group and time point) and germ-free mice (panel C; n = 3–15 per group and time point) following inoculation with 1010 (red squares) and 104 (blue circles) CFU/mouse, respectively. (D) Early colonization in mice (n = 4 per group) sampled every hour during the first 12 hours after gavage. Fitted exponential curves were used to extrapolate the time to reach levels of 2.5x109 CFU/g in the animals inoculated with 104 CFU. The average of these 4 values is represented by the vertical dotted line (at 15.5 h). (E-H) Representative fluorescent microscopy images of distal colon cross sections of SPF (E and G) and germ-free (F and H) mice infected with 1010 (E and F) and 104 (G and H) CFU/mouse of C. rodentium analyzed on day 7 post infection. All individual mice depicted were infected with a 1:1 mixture of bacteria carrying a mCherry (red) or GFP (green) fluorescent protein expression plasmid. Grey, F-actin stained with phalloidin; green, GFP-expressing C. rodentium; red, mCherry-expressing C. rodentium. Inset indicates area shown in higher magnification panel. Scale bars: 100 μm. (I) Numbers of A/E microcolonies in the distal colon of SPF mice infected with either 104 (blue circles) or 1010 (red squares) CFU of C. rodentium quantified over a time course of 10 days (n = 3–6 per group and time point, data pooled from 2 independent experiments). Connecting lines indicate means. (J) Numbers of A/E microcolonies in the distal colon of germ-free mice infected with either 104 (blue circles) or 1010 (red squares) CFU of C. rodentium quantified over a time course of 22 days (n = 2–6 per group and time point, data pooled from 3 independent experiments). Connecting lines connect means; horizontal dotted lines indicate detection limit; ****, p < 0.0001 (Student`s t-test); F-test, Fisher’s test.
Fig 2.
High-dose C. rodentium infection is associated with reduced severity of disease in germ-free wild-type mice.
(A) Body weight of germ-free mice infected with either 104 (blue circles) or 1010 (red squares) CFU/mouse of C. rodentium, (n = 15 until day 10). (B) Survival curve of the animals shown in A. (C) Splenic bacterial loads of mice shown in A infected with 104 (blue circles) or 1010 (red squares) CFU/mouse of C. rodentium for 10–13 days; n = 10–12 per group. (D) Histopathological scores of mice shown in panel A (Endpoint criterion: weight loss of >20 %). Mice were scored for epithelial hyperplasia and integrity, infiltration of PMNs, submucosal edema, and loss of goblet cells. Graph shows combined score (= sum of 5 individual scores). (E) Representative images of colonic histopathology scored in panel D (H&E staining) of mice infected for 13 days with 104 (top) and 1010 (bottom) CFU. Arrows in panel D indicate the individuals depicted. Scale bar: 100 μm; Sm, submucosa; Cr, crypts; Ep, epithelium; Lp, lamina proria; (F) Lipocalin-2 measurement by ELISA in feces from infected mice (n = 5 per group, same animals as shown in A; after day 13 n = 3 survivors in 104 group). Error bars indicate standard deviation. Dotted lines indicate detection limit; ns, statistically not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; statistical tests: Student`s t-test (A), Mantel-Cox test (B), Mann-Whitney U test (C, D, F).
Fig 3.
High-dose C. rodentium-infected MyD88/Trif-deficient mice do not display persistent disease attenuation.
(A-C) Germ-free MyD88-/-Triflps/lps mice were infected with either a low (104 CFU, blue dots) or a high dose (1010 CFU, red squares) of wild type C. rodentium. (A) qPCR quantification of bacterial ler mRNA in colon content of infected MyD88-/-Triflps/lps germ-free mice at time point 18 h. Expression of ler was normalized to the mRNA encoding bacterial housekeeping gene rpoD. (B) Microcolonies were quantitated in the distal colon after 1, 2, or 3 days of infection. (n = 3 per group, pooled from two independent experiments). (C) Body weights of germ-free MyD88-/-Triflps/lps mice infected with either a low (104 CFU, blue circles) or a high dose (1010 CFU, red squares) of wild type C. rodentium were monitored daily until reaching ethical endpoint. Symbols represent individual mice. (D) Representative fluorescent microscopy images of distal colon of germ-free MyD88-/-Triflps/lps mice after infection with doses of 104 or 1010 CFU of C. rodentium for 2 days (left) and 3 days (right). Every individual depicted was infected with a 1:1 mixture of bacteria carrying a mCherry and GFP expression plasmid, respectively. Grey, F-actin/phalloidin; green, GFP-expressing C. rodentium; red, mCherry-expressing C. rodentium. Scale bars: 200 μm and 100 μm as indicated. ns, statistically not significant; *, p < 0.05; Student`s t-test.
Fig 4.
An early window of opportunity for A/E microcolony induction in wild-type mice.
(A) Colonic luminal colonization trajectories of the pre-infection strain (red open symbols: circles, 104 CFU Wild type C. rodentium [WT]; squares, 1010 CFU WT; inverted triangles; 1010 CFU Δler mutant), the super-infection strain (blue filled symbols: circles, 104 CFU WT; squares, 1010 CFU WT), and total bacteria (pre-infection + super-infection strain; black triangles). Connecting lines connect means; error bars indicate standard deviations. Horizontal dotted lines indicate the lower detection limit. (B) Microcolony formation of superinfection strain after 7 days. N = 6–7 per group, pooled from 3 independent experiments. (C-G) Representative confocal fluorescent microscopy images of distal colon of mice shown in panel B. Grey, F-actin/phalloidin; green, anti-O-antigen Antibody (C. rodentium total); red, mCherry-expressing C. rodentium (pre-infection strain only); Scale bars: 200 μm and 100 μm as indicated; White squares indicate origin of higher-magnification image; *, p < 0.05; **, p < 0.01; ***, P < 0.001; ns, not significant (p ≥ 0.05); statistical test: one-way ANOVA.
Fig 5.
Early C. rodentium-induced innate immune response.
(A) Lipocalin-2 quantification by ELISA in feces of germ-free mice infected for 18 h with 104 CFU C. rodentium (blue circles), 1010 CFU C. rodentium (red squares), 1010 CFU C. rodentium Δler (green triangles), 1010 E. coli HS (orange hexagons) or left uninfected (grey diamonds). MyD88-/-Triflps/lps germ-free mice were infected for 18 h with 104 CFU C. rodentium (open blue circles), 1010 CFU C. rodentium (open red squares), or left uninfected (open grey diamonds). N = 4–13 per group. Data were pooled from 3 independent experiments. (B and C) Gene expression levels of Cxcl1 (B) and Nos2 (C) in the distal colon of infected mice as determined by qPCR. (D-G) Cytokine protein levels in the distal colon of infected mice as determined by Luminex technology. (H and I) Leukocyte analysis by flow cytometry of peripheral blood. Monocytes were defined as CD45+, CD11b+, CD115+ population (H). Neutrophils were defined as CD45+, CD11b+, Ly6G+ population. Quantities are expressed as absolute numbers of cells per mL blood. (J and K) Large intestinal leukocyte analysis by flow cytometry. Infiltrating monocytes were defined as CD45+, CD11b+, CD64-, LygG-, Ly6Chigh, and MHCII- population (J). Neutrophils were defined as CD45+, CD11b+, Ly6G+ population (K). Quantities are expressed as percentages of leukocytes (of CD45+ population). Dotted lines represent detection limit. ns, statistically not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; groups were compared with the matching uninfected control group; statistical tests: Kruskal-Wallis (A), and 1-way ANOVA (B-K).
Fig 6.
Ongoing A/E lesion induction in mice deficient for innate signaling through MyD88 and Trif.
(A) Colonic colonization dynamics in MyD88-/-Triflps/lps germ-free mice consecutively infected, first with mCherry+ KanR TetS C. rodentium wild type (WT) or an isogenic Δler mutant (1010 CFU, red squares and triangles), and 18 hours later superinfected with mCherry- KanS TetR C. rodentium wild type (104 CFU, blue circles). Total C. rodentium counts are depicted as black symbols. Connecting lines connect means; error bars indicate standard deviation; horizontal dotted lines indicate detection limit. (B and C) Representative fluorescent microscopy images of distal colon of MyD88-/-Triflps/lps mice shown in panel A pre-infected with wild type (B) and Δler (C) C. rodentium, respectively. Grey, F-actin/phalloidin; green, anti-Citrobacter O-antigen antibody (pre-infection strain and superinfecting strain); red, mCherry-expressing C. rodentium (pre-infection strain only; none detectable). Scale bars: 50 μm.