Fig 1.
AIEC modifies disease outcome following acute gastroenteritis.
(A). Infection scheme: NRAMP+ 129e mice were colonized with 2 x 109 cfu of AIEC strain NRG857c for 14 days to establish chronic colonization and low-grade intestinal inflammation (Phase 1). Control mice remained AIEC-naïve. AIEC-colonized and control mice were exposed to acute infectious gastroenteritis with 0.8 x 108 cfu of Salmonella enterica serovar Typhimurium (Phase 2). (B) Kaplan-Meier survival plots of 129e mice after Salmonella infection. n = 32 mice per group from 6 independent experiments (p<0.0001, log rank). (C) Percent change in body weight was monitored up to 27 days post-Salmonella infection. Data is expressed as a mean ± SEM of 5 mice per group from 8 independent experiments.
Fig 2.
Prior AIEC colonization worsens pathology following S. Typhimurium infection.
(A) Gross pathology in the small and large intestine of 129e mice 5 days after Salmonella infection. (B) H&E-stained cecal sections from 4 experimental groups of 129e mice (as indicated). Images are representative of 5 mice per group from 4 independent experiments. Arrows indicate submucosa edema, desquamation, epithelial sloughing, crypt hyperplasia, and inflammatory cellular infiltrates in the lumen and mucosa. Original magnification 40x. (C) Cecal pathology was scored from H&E stained sections taken from day 5. Data are the mean with ± SEM for 5 views per mouse and 5 mice per group. ***p<0.001 (Mann-Whitney). (D) Nitrite concentration in cecal samples on day 5 after Salmonella infection. Treatment groups are indicated. Data are the means with SEM. **p<0.01, ***p<0.001 (Mann-Whitney). (E) FITC-dextran concentration in the serum following oral gavage in the indicated groups. Data are the means with SEM. **p<0.01, ***p<0.001 (Mann-Whitney).
Fig 3.
Tissue-associated expansion of AIEC following Salmonella gastroenteritis.
(A) Fecal Salmonella loads on day 2, 4, and 5 after infection. Each data point is from one animal and represents 3–4 independent experiments. (B) Tissue-associated Salmonella burdens in the cecum, ileum, and spleen on day 5 after Salmonella infection. Each point is from one animal and data represents 3–4 independent experiments. (C) Tissue-associated AIEC burden in feces, cecum and ileum on day 5 after Salmonella infection. Each point is from one animal and data represent 3–4 independent experiments. (D) Tissue-associated E. coli K12 in feces, cecum and ileum on day 5 after Salmonella infection. Each point is from one animal. ns not significantly different between comparison groups, *p<0.05, **p<0.01, ***p< 0.001 (Mann-Whitney test).
Fig 4.
Prior AIEC colonization leads to heightened cellular and proinflammatory cytokine responses.
(A) Immunohistochemical staining of cecal tissue for F4/80+, GR1+, and CD3+ cells. Each image is representative of 2 independent experiments with 5 mice per group. Original magnification 200x. (B) Quantification of immunohistochemical staining. Data is from 6–8 views per section from 5 mice. *p<0.05, **p<0.01, ***p<0.001(Mann-Whitney test). (C) TNF-α, (D) IL-17 and (E) chemokines were measured using ELISA from supernatants after overnight incubation of explanted ceca. Data are the means ± SEM of 5 mice per group from 3 separate experiments. *p<0.05, **p<0.01, ***p<0.001 (one way ANOVA with Tukey for comparisons between groups).
Fig 5.
Resistance to host defense peptides is required for AIEC expansion in the inflamed gut and immunopathology.
129e mice were colonized with wild type AIEC or the peptide-sensitive ΔPI-6 mutant and then infected with Salmonella. Survival was assessed over 35 days and the tissue burden of AIEC was determined in the cecum (A) and the ileum (B). Each point represents one animal and the data represents 3 independent experiments. ***p< 0.001 (Mann-Whitney test). 129e mice were colonized with AIEC and then infected with either wild type Salmonella or a mutant that is less proinflammatory, S.tmavir. Tissue burden of AIEC (blue circles) and Salmonella (yellow circles) was determined in the cecum (C) and the ileum (D). Each point represents one animal and the data represents 2 independent experiments. *p<0.05; **p<0.01; ***p< 0.001 (Mann-Whitney test). (E) H&E-stained tissue samples from cecal tips are shown for the indicated infection groups. Original magnification 200x. (F) Quantification of histopathology from part E. Data are the means ± SEM of 5 mice per group from 2–3 separate experiments and 5 views per mouse. ***p<0.001 (one way ANOVA with Tukey). (G) TNFα levels determined from explanted cecal supernatants by ELISA. (H) Fecal lipocalin-2 levels determined from fecal pellets on day 5 after Salmonella infection. Data are means with SEM from 2–3 experiments. **p<0.01, ***p< 0.001 (Mann-Whitney test).
Fig 6.
Infectious colitis by C. rodentium prevents convalescence from AIEC colonization.
(A) Infection scheme. C57BL/6 mice colonized with 2 x 109 cfu AIEC NRG857c for 7 days or treated with PBS and then infected with 1 x 108 cfu C. rodentium or given PBS. (B) Fecal shedding of C. rodentium was monitored for 21 days. Data are means ± SEM of at least 5 mice per group/time point from 3 independent experiments. (C) Fecal shedding of AIEC after C. rodentium infection was monitored for 36 days. Data are means ± SEM of at least 5 mice per group/time point from 3 independent experiments. Tissue- associated AIEC was measured in the cecum (D) and colon (E) on day 14 and 21 after C. rodentium infection. Data are means ± SEM of at least 5 mice per group/time point from 3 independent experiments. **p<0.01 and ***p<0.001 (Mann-Whitney test).
Fig 7.
Mucosal epithelial restitution is delayed by AIEC following infectious colitis.
(A) H&E staining of colonic sections from C57BL/6 mice infected as indicated, on day 14 and day 21 after C. rodentium infection. Images are representative of 2 experiments with 5 mice per group per time point. Arrows indicate submucosa edema, desquamation, hyperplasia, inflammatory cellular infiltrates and epithelial sloughing. Original magnification 200x. Quantification of colonic pathology on day 14 (B) and day 21 (C) after C. rodentium infection. Measurements represent an average of at least 5 views per section and are the means with SEM from 5 mice. Quantification of crypt length (D) and goblet cell numbers (E) on day 14 and day 21 after C. rodentium infection. Measurements are from at least 5 views per section. Data are expressed as the means ±SEM of 5 mice per group/time point from 2 separate experiments. *p<0.05, **p<0.01, and ***p<0.001 (one way ANOVA with Tukey).