Interleukin-1 receptor signaling protects mice from lethal intestinal damage caused by the attaching and effacing pathogen Citrobacter rodentium - PubMed (original) (raw)

Interleukin-1 receptor signaling protects mice from lethal intestinal damage caused by the attaching and effacing pathogen Citrobacter rodentium

Sarah L Lebeis et al. Infect Immun. 2009 Feb.

Abstract

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to the intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes a breach of the intestinal epithelial barrier, leading to colitis via a vigorous inflammatory response resulting in diarrhea and a protective antibody response that clears the pathogen. Here we show that interleukin-1 receptor (IL-1R) signaling protects mice following infection with C. rodentium. Upon infection, mice lacking the type I IL-1R exhibit increased mortality together with severe colitis characterized by intramural colonic bleeding and intestinal damage including gangrenous mucosal necrosis, phenotypes also evident in MyD88-deficient mice. However, unlike MyD88(-/-) mice, IL-1R(-/-) mice do not exhibit increased pathogen loads in the colon, delays in the recruitment of innate immune cells such as neutrophils, or defects in the capacity to replace damaged enterocytes. Further, we demonstrate that IL-1R(-/-) mice have an increased predisposition to intestinal damage caused by C. rodentium but not to that caused by chemical irritants, such as dextran sodium sulfate. Together, these data suggest that IL-1R signaling regulates the susceptibility of the intestinal epithelia to damage caused by C. rodentium.

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Figures

FIG. 1.

IL-18 is not necessary for survival, although it is associated with slightly reduced colonic pathology. (A) Survival curves of 6- to 10-week-old IL-18−/− mice (n = 6) and their C57BL/6 controls (n = 12) infected with 1.5 × 108 CFU of C. rodentium. (B) Pathology scores were assessed in colonic tissue from uninfected C57BL/6 or IL-18−/− mice or from infected C57BL/6 or IL-18−/− mice 3 or 7 days p.i. The numbers of mice for each group ranged from 8 to 12 for C57BL/6 mice and 5 to 7 for IL-18−/− mice. Uninf, uninfected. (C) Pathology scores for day 7 p.i. are shown in each category assessed, including bleeding, ragged epithelium (Epi.), GMN, hyperplasia, goblet cell loss, edema, and polymorphonuclear leukocyte infiltration (PMN Infil.), for C57BL/6 (n = 12) and IL-18−/− (n = 7) mice. (D) Various tissues were harvested aseptically from C57BL/6 (n = 12) and IL-18−/− (n = 7) mice 7 days p.i. C. rodentium CFU were quantitated as described in Materials and Methods. MLN, mesenteric lymph nodes. Error bars represent the standard deviations.

FIG. 2.

IL-1R signaling is necessary for survival and prevention of severe pathology following infection with C. rodentium. (A) Survival curves of 6- to 10-week-old IL-1R−/− mice (n = 9) and their C57BL/6 controls (n = 12) infected with 1.5 × 108 CFU of C. rodentium. (B) Colons from IL-1R−/− and C57BL/6 mice 7 days p.i. (C) Pathology scores for colonic tissues from uninfected (Uninf) C57BL/6 or IL-1R−/− mice or from infected C57BL/6 or IL-1R−/− mice were assessed 3 or 7 days p.i. The numbers of mice ranged from 8 to 12 for C57BL/6 mice and from 8 to 13 IL-1R−/− mice for each bar (*, statistical significance in comparison to uninfected C57BL/6 samples, P < 0.01; **, statistical significance in comparison to uninfected IL-1R−/− samples, P < 0.001; #, statistical significance between C57BL/6 and IL-1R−/− samples, P < 0.001). Error bars represent the standard deviations. (D and E) Colon supernatants from uninfected C57BL/6 mice or C57BL/6 mice 3 or 7 days p.i. were assessed for IL-6 (n = 9 to 12) and IFN-γ (n = 12 or 13) levels by ELISA. The difference between levels of cytokine production in uninfected mice and mice 3 days p.i. was significant at P < 0.02 for IL-6 and P < 0.01 for IFN-γ. Colon supernatants from uninfected IL-1R−/− mice, IL-1R−/− mice 3 days p.i., and IL-1R−/− mice 7 days p.i. were assessed also for IL-6 (n = 7 to 15) and IFN-γ (n = 7 to 13) levels by ELISA. No statistically significant difference was evident. Horizontal bars indicate sample means.

FIG. 3.

IL-1R signaling is required to prevent increased colonic damage and specifically GMN following infection with C. rodentium. (A) H&E staining of colonic tissue from an uninfected C57BL/6 mouse. (B) H&E staining of colonic tissue from a C57BL/6 mouse 7 days p.i. (C) Pathology scores of the damage measurements, including bleeding, ragged epithelium (Epi.), and GMN, were assessed in colonic tissue from C57BL/6 (n = 12) or IL-1R−/− (n = 13) mice 7 days p.i. (*, statistical significance in comparison to C57BL/6 samples, P < 0.001; **, statistical significance in comparison to C57BL/6 samples, P < 0.01). (D) H&E staining of uninfected colonic tissue from an IL-1R−/− mouse. (E) H&E staining of IL-1R−/− colonic tissue 7 days p.i. Magnification, ×400 for all H&E images. (F) C. rodentium was grown as standing cultures for up to 8 h in LB without supplement or in LB supplemented with 3.5 mg/ml serum or with 3.5 mg/ml RBC lysate, and OD600 was measured. OD600 differences were evident in cultures supplemented with RBC lysate by 4 h p.i. and with serum by 6 h p.i. Each sample (n = 6) was blanked to an uninfected sample of the media (*, statistical significance in comparison to LB samples, P < 0.05; **, statistical significance in comparison to LB samples, P < 0.02; #, statistical significance in comparison to LB samples, P < 0.01). (G) C. rodentium was incubated in LB without supplement or supplemented with 3.5 mg/ml serum or with 3.5 mg/ml RBC lysate for 6 h, and the number of bacteria in cultures was determined as described in Materials and Methods. Data presented are from a representative experiment. The experiment was repeated twice with three replicates each. Error bars represent the standard deviations. (H) Intestinal permeability was measured by the ability of FITC-dextran to migrate from the intestine to serum in C57BL/6 or IL-1R−/− mice that were left uninfected (Uninf) or infected with C. rodentium. Serum was harvested 7 days p.i. Infected IL-1R−/− mice displayed a significant increase in intestinal permeability compared to that for uninfected controls, and no changes in permeability were evident in infected or uninfected C57BL/6 mice (*, statistical significance in comparison to uninfected levels, P < 0.01; **, statistical significance in comparison to wild-type levels at 7 days p.i., P < 0.01).

FIG. 4.

IL-1R−/− mice have C. rodentium loads and neutrophil responses equivalent to those seen in C57BL/6 mice. (A) Various tissues were harvested aseptically from C57BL/6 (n = 12) and IL-1R−/− (n = 9) mice 7 days p.i. C. rodentium CFU were quantitated as described in Materials and Methods. MLN, mesenteric lymph nodes. (B) Pathology scores for recruitment measurements, including edema and polymorphonuclear leukocyte infiltration (PMN Infil.), were assessed in colonic tissue from C57BL/6 (n = 12) or IL-1R−/− (n = 13) mice 7 days p.i. (*, statistical significance in comparison to C57BL/6 samples, P < 0.01). (C) Manual neutrophil counts in crypts from C57BL/6 (n = 5 to 15) and IL-1R−/− (n = 7 to 15) colons (*, statistical significance in comparison to uninfected [uninf] C57BL/6 samples, P < 0.01; **, statistical significance in comparison to uninfected IL-1R−/− samples, P < 0.02; #, statistical significance in comparison to uninfected samples, P < 0.001). (D) CFU (5 × 105) of C. rodentium incubated with neutrophils (2.5 × 105) from C57BL/6 or IL-1R−/− mice were killed with equal efficacies. Results are from a representative experiment repeated three times in triplicate. Error bars represent the standard deviations.

FIG. 5.

IL-1R−/− and C57BL/6 mice have equal levels of hyperplasia following infection with C. rodentium. (A) The pathology scores were assessed for the degrees of hyperplasia in colonic tissue taken from IL-1R−/− (n = 13) and C57BL/6 (n = 12) mice. (B) Crypt lengths of colonic tissue from uninfected (uninf) C57BL/6 or IL-1R−/− mice or from C57BL/6 or IL-1R−/− mice 3 or 7 days p.i. were measured (*, statistical significance in comparison to uninfected samples, P < 0.001). Error bars represent the standard deviations.

FIG. 6.

IL-1R−/− mice suffer from increased pathology, particularly hyperplasia, following DSS administration. (A) Pathology scores were assessed in colonic tissue from untreated C57BL/6 or IL-1R−/− mice or from treated C57BL/6 or IL-1R−/− mice 7 or 14 days posttreatment. The numbers ranged from 6 to 10 C57BL/6 mice and 9 to 12 IL-1R−/− mice for each bar (*, statistical significance in comparison to samples from C57BL/6 mice 14 days posttreatment, P < 0.01). (B) Pathology scores for each category were assessed in colonic tissue from treated C57BL/6 or IL-1R−/− mice 14 days posttreatment. Epi., epithelium; PMN Infil., polymorphonuclear leukocyte infiltration. (C) Crypt lengths of colonic tissue from untreated C57BL/6 or IL-1R−/− mice or from C57BL/6 or IL-1R−/− mice 7 or 14 days posttreatment were measured (*, statistical significance in comparison to uninfected samples, P < 0.01; **, statistical significance in comparison to uninfected samples, P < 0.001; #, statistical significance between C57BL/6 and IL-1R−/− samples, P < 0.02; ^, statistical significance between C57BL/6 and IL-1R−/− samples, P < 0.01). Error bars represent the standard deviations.

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Interleukin-1 receptor signaling protects mice from lethal intestinal damage caused by the attaching and effacing pathogen Citrobacter rodentium - PubMed (original) (raw)