Amelioration of Clostridium difficile Infection in Mice by Dietary Supplementation With Indole-3-carbinol

Abstract

Objective: To determine the therapeutic effects of dietary supplementation on Clostridium difficile infection (CDI).

Background: With limited treatment options, the rise of C. difficile-associated disease has spurred on the search for novel therapies. Recent data define a role for the aryl hydrocarbon receptor (AHR) and diet-derived AHR ligands in mucosal immunity. We investigated the efficacy of indole-3-carbinol (I3C), a dietary supplement, and AHR precursor ligand in a murine model of CDI.

Methods: C57BL/6 (B6), AHR, and AHR mice were placed on either grain-based or semipurified diets with or without I3C before and during CDI. Mice were followed clinically for a minimum of 6 days or euthanized between days 0 and 4 of inoculation for analysis of the inflammatory response and microbiota.

Results: B6 mice fed an AHR ligand-deficient, semipurified diet have significantly increased disease severity (P<0.001) and mortality (P < 0.001) compared with mice fed on diet containing I3C. The addition of I3C to the diet of AHR null mice had less of an impact than in AHR heterozygous littermates, although some protection was seen. Mice on semipurified I3C-diet had increased cecal Tregs, ILC3s, and γδ T cells and an increased neutrophilic response without increased inflammation or bacterial translocation compared with controls.

Conclusions: I3C is a powerful treatment to reduce impact of CDI in mice. The findings indicate I3C may be acting through both AHR-dependent and -independent mechanisms in this model. Dietary supplementation with I3C is a potential new therapy for prevention and amelioration of C. difficile disease.

Figure 1. Addition of dietary I3C reduces CDI-related mortality and morbidity

(A) Diagram of CDI model testing efficacy of dietary I3C on disease progression. Treatments include addition of antibiotics (ABx) to drinking water, i.p injection of Clindamycin (CLI) and oral gavage with C. difficile spores. (B) Percent survival, (C) mean percent weight +/− SEM and (D) mean clinical score +/− SEM after inoculation with 10⁵ VPI 10463 C. difficile of male B6 mice fed standard chow or chow + I3C (1000 ppm) starting two weeks prior to ABx treatment. Data was pooled from two independent experiments. (E) Percent survival, (F) mean percent weight +/− SEM and (G) mean clinical score +/− SEM after inoculation with 10⁴ VPI 10463 C. difficile of male B6 mice fed semi-purified diet (diet) or semi-purified diet supplemented with I3C (1000 ppm) starting two weeks prior to ABx treatment. Data was pooled from three independent experiments. Daily mice at risk are indicated in panels B and F. Log-rank test was used for survival analysis, two-tailed Student’s t-test was used for comparing daily percent weight and Mann-Whitney U test was used for comparing daily clinical score. * P ≤ 0.05; ** P ≤ 0.01; *** P < 0.001.

Figure 2. Protective effects of dietary I3C are both AHR dependent and independent

(A) Percent survival, (B) mean percent weight +/− SEM and (C) mean clinical score +/− SEM after inoculation with 10⁵ VPI 10463 C. difficile of male AHR ^+/− and AHR^−/− mice fed standard chow. Data was pooled from two independent experiments. (D,G) Percent survival, (E,H) mean percent weight +/− SEM and (F,I) mean clinical score +/− SEM after inoculation with 10⁴ VPI 10463 C. difficile of male AHR^+/− (D – F) or male AHR^−/− (G – I) mice fed semi-purified diet (Diet) or semi-purified diet supplemented with I3C (Diet + I3C) starting two weeks prior to ABx treatment. Data was pooled from two independent experiments. Daily mice at risk are indicated in panels B, E, H. Log-rank test was used for survival analysis, two-tailed Student’s t-test was used for comparing daily percent weight and Mann-Whitney U test was used for comparing daily clinical score. * P ≤ 0.05; ** P ≤ 0.01.

Figure 3. Lack of dietary AHR ligands alters gut immune cells prior to inoculation with C. difficile

(A and B) Relative Cyp1a1 and FoxP3 expression as measured by RT-PCR in semi-purified diet and I3C mice compared to levels of β-actin. (C) Representative plots of isolated cecal cells with percent parent population. (D–F) Graphs of semi-purified diet and I3C mice at day 0 demonstrating frequency of cLP Tregs (Live/CD45⁺/CD11b⁻/Ly6G⁻/CD3⁺/CD4⁺/FoxP3⁺; D), ILC3s (Live/CD45⁺/CD11b⁻/Ly6G⁻/CD3⁻/CD4⁻/RORγt⁺; E), and number of IEL γδ T cells (Live/CD45⁺/CD11b⁻/Ly6G⁻/TCRβ⁻/CD3⁺/ TCRγδ⁺; F). All data are representative of n = 4 per group, with error bars showing SEM, repeated in 2 independent experiments. * P ≤ 0.05; ** P ≤ 0.01. Two-tailed Student’s t-test or Mann-Whitney U test used for comparing averages.

Figure 4. Mice fed I3C supplemented diet display increased neutrophil response to C. difficile without increased cecal inflammation

(A) Cecal C. difficile CFU/g feces in diet and diet + I3C mice at day 2 and 4 (n=4 per group). Limit of detection indicated by dash-line. Determination of the presence or absence of C. difficile cfu for each day determined by Chi-square analysis. (B and C) Representative flow plots and % of cLP neutrophils per CD45+ cells on day 0 (n=4 per group) and day 3 (n=8 per group) after inoculation in diet and diet + I3C mice. (D and E) Representative histology at 100x and 400x in semi-purified diet and I3C mice at day 3 (arrows depict neutrophils) and corresponding histopathological scoring (n=10 per group). (F) Relative IL-22, IL-17, and IFN-γ expression at day 0 and 4 as measured by RT-PCR in semi-purified diet and diet + I3C mice normalized to β-actin (n=4–10 per group). (G) Measurement of total IL-22 and IL-17A protein content per cecum by ELISA.

Figure 5. I3C added to the diet reduces bacterial translocation during CDI

Bacterial counts from tissue homogenates of (A) spleen, (B) lung, (C) liver and (D) kidney on day 4 following CDI in mice fed diet or diet + I3C. (E) Survival, (F) weight loss, and (G) clinical scores of mice on semi-purified diet treated with plain drinking water or drinking water with 0.125 mg/mL ciprofloxacin beginning 1 day after inoculation with 10⁴ VPI 10463 C. difficile (n = 10 per group). All error bars show SEM. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Log-rank test used for survival analysis and two-tailed Student’s t-test or Mann-Whitney U test used for comparing averages.