Enteropathogenic and enterohemorrhagic Escherichia coli
(EPEC and EHEC) are some of the most common pathogenic bacteria that contribute to clinical infections in both animals and humans [1
]. EPEC/EHEC exist as normal microbes in the gastrointestinal tract of ruminant animals, such as cow and sheep, but are asymptomatic, while causing severe disease in humans [2
]. The symptoms in humans include abdominal cramps, vomiting, and/or diarrhea, which may progress to hemorrhagic colitis [3
]. About 30% of confirmed cases require hospitalization, and about 10% of cases develop into hemolytic uremic syndrome (HUS) that is characterized by anemia, kidney failure, and low platelet counts [5
]. Antibiotics are commonly prescribed for bacterial infections but their overuse on farms and in hospitals has increased the incidence of multidrug resistant (MDR) E. coli
infections, which have become a significant threat to human health [7
]. The development of alternative strategies for controlling the spread and treatment of EPEC and EHEC are necessary to protect public health and minimize the economic costs associated with outbreaks of these bacteria.
The study of EPEC and EHEC is limited by the inability of these bacteria to mimic human infections and disease when using a rodent model. Citrobacter rodentium
(Cr) is a gram negative microbe that naturally infects mice, causes diarrhea and shares 67% of its genes with EPEC and EHEC, including genes associated with pathogenicity and virulence [1
]. Cr infection in mice causes attaching and effacing (A/E) lesions and a potent TH1/TH17 inflammatory response similar to those observed in human EPEC/EHEC infections. Therefore, it has become a standard small-animal model to study infectious colitis [8
]. Cr infection results in various changes to the colon of mice, including epithelial cell proliferation, crypt hyperplasia, an uneven apical enterocyte surface, crypt dilation, and mucosal thickening [10
]. Except in highly susceptible mouse strains such as C3H/HeN, colonization of Cr is limited to the colon, with few bacteria reaching systemic organs and the bloodstream [11
]. Following oral administration, Cr initially colonizes the cecal patch and then migrates to the colon by day 3 post-infection. Bacterial load in the distal colon peaks by day 7 and remains at high levels through day 12, and is typically cleared by day 21 [13
]. In general, Cr infection can serve as a useful model for studying compounds that may prevent or mitigate the effects of EPEC or EHEC infections.
Indole-3-carbinol (I3C) is a dietary compound (Figure 1
) derived from glucobrassicin, a glucosinolate found in cruciferous vegetables cruciferous vegetables such as broccoli, cabbage and cauliflower. The concentration of I3C from cruciferous vegetables can be inferred from the content of glucobrassicin which varies in different kinds of cruciferous vegetables, ranging from 0.24 μmol·g−1
DW to 6.2 μmol·g−1
]. I3C and its derivatives have attracted increased attention due to their important role as anti-inflammatory, anti-tumor and immune modulating agents [16
]. Substantial evidence indicates that the anti-inflammatory and anti-cancer effects of I3C and its metabolic derivatives are attributed to their ability to modulate several nuclear transcriptional factors including the estrogen receptor (ER), nuclear factor-κB (NF-κB), and the aryl hydrocarbon receptor (AhR), which contribute to maintaining hormonal homeostasis, inhibiting cell cycle progression/apoptosis, inducing DNA repair, and enhancing carcinogen metabolism [17
]. Although the potential value of I3C and its derivatives in cancer prevention and therapy are well known, the exact underlying mechanisms are still unclear.
Infection-induced inflammation is a common risk factor for certain types of cancer. Cr infection induces both innate and adaptive immunity, involving the recruitment of immune cells and the release of multiple cytokines and antimicrobial peptides that, are required for clearance of Cr [19
]. It is well known that IL-22 producing CD4+
T cells are essential for controlling Cr infection; however, the T cell-derived cytokines (IL17A, IFN-γ and TNF-α) contribute to intestinal tissue injury either directly or indirectly [22
]. In addition to CD4+
T cells, the group 3 innate lymphoid cells (ILC3s) also produce IL22, which is crucial for clearance of Cr [24
]. AhR-deficient mice lack IL-22-producing ILC3 in the intestinal lamina propria, and have increased mortality when infected with Cr, suggesting that AhR-dependent signal pathways are important in controlling and clearing Cr [25
]. I3C is a powerful AhR activator, which helped suppress Cr-induced inflammation by enhancing AhR activation [26
]. Although the protective effect of I3C against Cr infection is known, little information is available related to effects of I3C on the immune response to Cr infection in Cr-susceptible mice. Hence, further study of the mechanism of action of I3C and/or cruciferous vegetable consumption on the immune and inflammatory responses to Cr infection is warranted.
To address these questions, we focused on characterizing the effects of dietary I3C on the immune response to Cr infection in inflammation-susceptible mice (C3H/HeN strain) to gain further insight into the immunomodulatory properties of dietary I3C. We found the protective effects of dietary I3C against Cr infection occur by down regulating the proinflammatory response while maintaining IL-22 production.
2. Materials and Methods
2.1. Animals and Diet
C3H/HeN and C57BL/J6 (5-week-old male) mice were purchased from Charles River (Frederick, MD). Mice were housed in ventilated filter-top cages at the USDA BHNRC animal facility under 12-h light/dark cycle. One week of acclimation on chow diet was conducted prior to the dietary treatments. Mice were then, randomized into four experimental groups (n = 8 per group): (1) Uninfected mice on control diet, (2) infected mice on control diet, (3) uninfected mice on treatment diet, and (4) infected mice on treatment diet. Mice were treated for two weeks prior to Cr infection and remained on their respective diets until the end of the experiment. Body weights and food consumption were recorded weekly. All experiments were approved by the USDA-ARS Beltsville Institutional Animal Care and Use Committee (18-027).
Mice were fed an AIN-93M diet with or without 1 µmol I3C/g diet. I3C was purchased from Sigma Chemical Company (St. Louis, MO, USA). A dose of 1 µmol I3C/g diet (147 mg/kg) in mice is roughly equivalent to a dose of 11.4 mg/kg in average adult human. In a clinical study, consumption of I3C was tolerated in doses up to 1200 mg/day in male and female cancer patients. Our selected concentration of I3C for this study was in the range achievable through dietary consumption as well as in a low dose chemo-preventive range [27
2.2. Cr Infection
Mice were infected with Cr using established protocols [26
]. The Cr strain used in this study was a nalidixic acid-resistant mutant of strain DBS100 (ATCC 51459). A frozen stock of Cr was streaked out on a Luria-Bertani (LB) agar plate and grown overnight at 37 °C. An overnight LB culture grown at 37 °C was started by picking one well-isolated colony. The culture was then expanded and grown to an OD600 nm of ≈1.5, the bacteria were collected and re-suspended in LB medium to a concentration of 1.25 × 1010
CFU/mL. After fasting 4–6 h, mice were infected by oral gavage with 0.2 mL of the bacterial suspension (2.5 × 109
CFU). To serve as uninfected controls mice on either diet were given only LB medium. The dose was confirmed by retrospective plating on LB agar plates containing 50 µg/mL nalidixic acid.
2.3. Sample Collection
On days 4, 7, 11, 14, 17, and 20 post-infection, fresh fecal pellets of mice were collected for determining the Cr load in feces. The pellets were homogenized in LB broth, serially diluted, and then plated on LB/agar plates containing 50 μg/mL nalidixic acid, incubated at 37 °C overnight. The colonies were enumerated the following day.
Mice were weighed and then euthanized on day 12 and 21 after infection. Blood samples were collected, the serum separated and stored at −80 °C. The spleen, cecum and the distal 5 cm of colon (without fecal pellets) were removed aseptically and weighed. 1cm portions of the distal colon were fixed in 4% formalin for histology or snap frozen in liquid nitrogen for gene expression analysis. The remainder of colon was homogenized and used to measure the tissue Cr load by plating serial dilutions on LB/agar plates containing 50 μg/mL nalidixic acid. Results are expressed as CFUs per gram of colon.
2.4. Histological Analysis
Approximately 1 cm sections of distal colon tissue in each group of mice were fixed in 4% formalin and embedded in paraffin, 5-μm sections were cut and stained with hematoxylin and eosin (H&E). The histological grading of coded sections was evaluated for the degree of edema (0–3), surface of epithelium (0–4), loss of crypt architecture (0–4), degree of hemorrhaging (0–4), and the presence of an inflammatory cell infiltrate (0–4). Crypt depth was measured using a Nikon Eclipse E800 microscope and Nikon NIS-Elements software V4.6. Only well-oriented crypts were measured, and 12 or more individual measurements were averaged for each mouse.
2.5. Gene Expression Analysis
To determine the gene expression in spleen and colon samples, total RNA was harvested from splenic and colonic tissue using RNeasy Mini kit (Qiagen, Valencia, CA, USA) and TRIzol reagent (Life Technology, NY, USA), respectively. The concentration and integrity of RNA were measured using a Bioanalyzer (Agilent 2100 Bioanalyzer, Santa Clara, CA, USA). RNA with an integrity number above 8 was used for real-time qRT-PCR. The Affinity Script Multi-temperature cDNA Synthesis kit from Agilent was used to reverse-transcribe mRNA to complementary DNA. Real-time PCR was performed on Applied Biosystems ViiA7 Real-Time PCR System using TaqMan®
Gene Expression Assay (Invitrogen, Carlsbad, CA, USA). To evaluate the effects of treatment, genes of interest were normalized to the housekeeping gene TATA box binding protein (Tbp) and analyzed using the ∆∆Ct method. The primers/probes for gene expression analysis were purchased from Life Technology are as follow (Table 1
2.6. Serum Cytokines Analysis
Serum cytokines profiles of mice (12 days post-infection) were assessed by using a Bio-Plex Pro™Mouse Cytokine 23-plex assay (Bio-Rad, Hercules, CA, USA) that was performed on a Luminex 200 system and Bioplex HTF in accordance to the manufacturer’s instructions. The results were analyzed using Bio-plex Manager™ software (Bio-Rad, Hercules, CA, USA).
2.7. Immunoglobulin Analysis
Nunc Maxi-Sorb plates (Corning, NY, USA) were coated with Cr antigen (10 μg/mL; 50 μL per well in 1X PBS), overnight at 4 °C. After washing with PBS mixed with Tween 20 0.05% (PBS-T), the plates were blocked with 3% nonfat dried milk in D-PBS (100 μL/well) for 2 h at room temperature. Plates were washed 3 times with PBS-T and then 50 μL of mouse serum (12 days post-infection) diluted (1:300 for IgG, M and 1:20 for IgA) in PBS-Tween was added to each well, incubate at 37 °C for 30 min. After incubation, the wells were washed 4X using PBS-T and 50 μL of 1:300 diluted Biotinylated anti-mouse IgG/IgM (Vector Laboratories, Burlingame, CA, USA) was added to each well. After 30 min of incubation at 37 °C, the plates were washed 4X with PBS-T and 50 μL/well streptavidin-HRP (1:1000 dilution from stock) (Vector Laboratories, Burlingame, CA, USA) was added. The plates were then incubated for 30 min at 37 °C, followed by the addition of TMB substrate (preheated at 37 °C, mix before using) for colorimetric detection. The reaction was stopped by adding 50 μL of 4N H2SO4 and the absorbance at 450 nm was measured immediately using a microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA). Data is expressed as either the OD450 nm or the ratio of infected OD450 nm/uninfected OD450 nm.
2.8. Statistcal Analysis
Results are expressed as the mean ± standard deviation (SD). Statistical analysis of this study was conducted by using GraphPad Prism 7 (2018, GraphPad Software, San Diego, CA, USA). Significance of differences between the mean of each group were analyzed using Student’s t test or one-way ANOVA followed by Fisher’s LSD test. In figures where more than two treatment groups are compared, groups with different letter are statistically significantly different (p value < 0.05).
infections in humans have become an increasing serious global public health issue due to emergent MDR E. coli
]. Developing sound strategies and means to inhibit E. coli
infections would be beneficial for promoting human health and containment of health care cost. I3C derived from cruciferous vegetables, exerting anti-microbial, anti-inflammatory, and anti-tumor properties [17
], may serve as an alternative to antibiotics for prevention and therapy of E. coli
infection. In the present study, the effects of dietary I3C consumption on Cr infection of Cr-sensitive C3H/HeN mice was examined as a model for human E. coli
infections. Our study demonstrated dietary consumption of I3C was able to reduce the pro-inflammatory response to a Cr infection and highlights the potential use of dietary regimes to help protect against a common intestinal infection. The amount of I3C administrated in these studies was in the range achievable through dietary consumption, and therefore, is applicable to humans [29
Mice infected with Cr often develop acute colitis accompanied by an overgrowth of Cr and self-limiting inflammation in murine intestinal lumen [10
]. Dietary I3C alleviated Cr-induced weight loss and suppressed colonic and splenic inflammation in C3H/HeN mice. However, there were no differences in colon and fecal bacteria load between the Cr-infected mice fed control or I3C diet, suggesting that the main effect of I3C consumption was on modulation of the host immune response rather than a direct effect on clearance of Cr. Our results showed that spleen appeared to be one of the main target organs that benefited from dietary I3C. The spleens of C3H/HeN mice were enlarged due to Cr infection and I3C attenuated the Cr-induced splenomegaly in mice. The spleen enlargement is probably due to an increase in macrophages as indicated by the increase in macrophage marker expression and macrophage-associated cytokines in infected control-fed mice compared to I3C-fed mice. This finding was consistent with Maaser et al. who found that the normally narrow marginal zone of spleen in mice was widened, with numerous macrophages, at 2 weeks after infection [36
]. In addition, CD4+
T cells and B cells are essential for the development of immunity and clearance of the pathogen [19
]. We found that Cr-infection appeared to lower expression of T-cell and B-cell markers in the spleen regardless of diet. These data support a depletion of these immune cells in the spleen in response to Cr infection. However, this effect was not modulated by dietary I3C. The decrease of spleen size in Cr-infected mice fed I3C correlates with the reduced levels of circulating pro-inflammatory cytokines and chemokines.
The colon is the primary tissue affected by Cr infection. The infection of mice with Cr induces a robust innate and adaptive mucosal immune responses, and causes various changes to the colon that include epithelial cell proliferation, crypt hyperplasia, crypt dilation, an uneven apical enterocyte surface, and mucosal thickening [22
]. Infected I3C-fed mice appeared to have similar colonic pathology as the infected control diet-fed mice. However, our data showed that dietary I3C significantly reduced the increase in the mucosal thickness in response to Cr infection on day 12 but not on day 21 post-infection. The I3C-induced reduction in mucosal thickness is associated with a decreased pro-inflammatory response in I3C-treated mice at day 12 post-infection. By day 21, the infection is essentially cleared in both control and I3C-treated mice. Substantial healing of the mucosa had occurred by day 21 and no differences mucosal thickness were observed between infected control and I3C-treated mice.
Most cytokine markers of Cr-induced inflammation were attenuated in colon tissue from Cr-infected mice on I3C diet compared to the infected mice on control diet, suggesting an anti-inflammatory effect of I3C on pathogen-induced acute colitis. The effects appeared to be selective as IL-22 mRNA levels that are closely associated with protection against Cr were unaffected by I3C treatment, while the cytokines associated with pathogenicity, such as IL17A, IL6, IL-1β, TNF-α, and IFN-γ, were decreased by feeding I3C. Furthermore, the anti-inflammatory effects of I3C appeared to be systemic as serum cytokines and chemokines markers (IL17, TNF-α, IL12 (p40, p70) and G-CSF) induced by Cr infection were all significantly attenuated in infected animals fed I3C diet.
In addition to the suppression of pro-inflammatory cytokines, the protective effects of I3C may be due, at least in part, to maintaining IL-22 levels. IL-22 was demonstrated to be protective against Cr infection and is a potent inducer of antimicrobial peptides, including β-Defensin, Lipocalin-2, RegIIIg and mucins [23
]. Many types of immune cell, such as CD4+
T cell and ILC3 cell, secrete the anti-inflammatory cytokine IL-22 [24
]. Production of IL-1β and IL-17A and the AhR receptor pathway have been reported to modulate the production of IL-22 [40
]. Additionally, I3C may stimulate IL-22 production via the gut microbiota [43
]. Given the systemic inhibitory effects of I3C consumption on IL-1β, IL-17A and its known action as an AhR agonist, it is likely that I3C is acting through AhR to maintain IL-22 expression and while repressing expression of pro-inflammatory cytokines. The precise target cells for I3C will require further validation.
The spleen has two critical functions in host defense against Cr infection, one is removing bacteria from bloodstream, another is producing antibodies for clearance of Cr [44
]. A possible mechanism that may explain effect of I3C on spleen and systemic immune responses is related to Cr-driven IgG production. IgG antibodies are required for mediating protective immune against Cr infection [36
]. Mice fed I3C seemed to elicit an enhanced Cr-reactive IgG response due to the Cr infection. The enhanced response may aid the removal of bacteria, and thus attenuate inflammation in spleen and the host’s immune system as a whole. The effect of I3C seems to be specific to IgG and not observed for IgM or IgA. Additionally, the effect of Cr-infection on immunoglobulin appeared to be very different between sensitive C3H/HeN mice and the resistant C57BL/J6. The resistant mice elicit relatively small immunoglobulin response upon Cr-infection and I3C-fed animals exhibited lower IgM levels. This observation is consistent with I3C preventing Cr attachment and growth in colonic tissue in resistant C57BL/J6 mice [26
One of our initial hypotheses was that I3C may act through CD8-dependent cytotoxic responses. However, our data indicates otherwise. Cr infection induced up-regulation of CD8+ T cell markers such as Cd8a, Cd8b, and FasL, suggesting involvement of CD8-mediated pathways in Cr infection. However, there were no difference in CD8-related markers between control-fed and I3C fed infected mice. Hence, the role of I3C appears relatively specific to its effect on cytokine and chemokine expression.