Dietary Indole-3-Carbinol Alleviated Spleen Enlargement, Enhanced Igg Response in C3H/Hen Mice Infected with Citrobacter Rodentium

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Article Dietary Indole-3-Carbinol Alleviated Spleen Enlargement, Enhanced IgG Response in C3H/HeN Mice Infected with Citrobacter rodentium

Yanbei Wu 1,2,3,4, Jing Wang 1 , Qiang He 4, Liangli Yu 3 , Quynhchi Pham 2, Lumei Cheung 2, Zhi Zhang 3, Young S. Kim 5, Allen D. Smith 2,* and Thomas T. Y. Wang 2,*

1 China-Canada Joint Lab of Food Nutrition and Health (Beijing), Beijing Technology & Business University, Beijing 100048, China; [email protected] (Y.W.); [email protected] (J.W.) 2 Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research Center, USDA-ARS, Beltsville, MD 20705, USA; [email protected] (Q.P.); [email protected] (L.C.) 3 Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA; [email protected] (L.Y.); [email protected] (Z.Z.) 4 College of Biomass Science and Engineering, Sichuan University, Chengdu 610065, China; [email protected] 5 Nutritional Science Research Group, Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] * Correspondence: [email protected] (A.D.S.); [email protected] (T.T.Y.W.); Tel.: +(301)-504-8577 (A.D.S.); +(301)-504-8459 (T.T.Y.W.) Received: 6 September 2020; Accepted: 11 October 2020; Published: 15 October 2020

Abstract: Enteropathogenic and enterohemorrhagic Escherichia coli are important enteric pathogens that induce hemorrhagic colitis or even fatal hemolytic uremic syndrome. Emerging evidence shows that some bio-actives derived from fruits and vegetables may serve as alternatives to antibiotics for overcoming multidrug resistant E. coli infections. In this study, the Citrobacter rodentium (Cr) infection model was utilized to mimic E. coli-induced acute intestinal inflammation, and the effects of a cruciferous vegetable-derived cancer protective compound, indole-3-carbinol (I3C), on the immune responses of Cr-susceptible C3H/HeN mice were investigated. Dietary I3C significantly inhibited the loss of body weight and the increase in spleen size in Cr infected mice. In addition, I3C treatment reduced the inflammatory response to Cr infection by maintaining anti-inflammatory cytokine IL-22 mRNA levels while reducing expression of other pro-inflammatory cytokines including IL17A, IL6, IL1β, TNF-α, and IFN-γ. Moreover, the serum cytokine levels of IL17, TNF-α, IL12p70, and G-CSF also were down-regulated by I3C in Cr-infected mice. Additionally, dietary I3C specifically enhanced the Cr-specific IgG response to Cr infection. In general, dietary I3C reduced the Cr-induced pro-inflammatory response in susceptible C3H/HeN mice and alleviated the physiological changes and tissue damage induced by Cr infection but not Cr colonization.

Keywords: indole-3-carbinol; C3H/HeN mice; Citrobacter rodentium; colitis; immunoregulation

1. Introduction 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 [3,4]. 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 [5,6].

Nutrients 2020, 12, 3148; doi:10.3390/nu12103148 www.mdpi.com/journal/nutrients Nutrients 2020, 12, 3148 2 of 15

Nutrients 2020, 12, x FOR PEER REVIEW 2 of 16

Antibioticsinto hemolytic are commonly uremic prescribedsyndrome (HUS) for bacterial that is infectionscharacterized but theirby anemia, overuse kidney on farms failure, and and in hospitalslow has increasedplatelet counts the incidence[5,6]. Antibiotics of multidrug are commonly resistant prescribed (MDR) forE. bacterial coli infections, infections which but their have overuse become a significanton farms threat and in to hospitals human healthhas increased [7]. The the development incidence of multidrug of alternative resistant strategies (MDR) E. for coli controlling infections, the spreadwhich and have treatment become ofa significant EPEC and threat EHEC to human are necessary health [7]. to The protect development public of health alternative and minimizestrategies the economicfor controlling costs associated the spread with and outbreaks treatment ofof theseEPEC bacteria.and EHEC are necessary to protect public health Theand minimize study of the EPEC economic and EHECcosts associ is limitedated with by outbreaks the inability of these of bacteria. these bacteria to mimic human infectionsThe and study disease of EPEC when and using EHEC a rodentis limited model. by theCitrobacter inability of rodentiumthese bacteria(Cr) to is mimic a gram human negative 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 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 EHEC, including genes associated with pathogenicity and virulence [1]. Cr infection in mice causes attachingattaching and and effacing effacing (A /(A/E)E) lesions lesions and and a a potent potent TH1TH1/TH17/TH17 inflammatory inflammatory response response similar similar to those to those observedobserved in human in human EPEC EPEC/EHEC/EHEC infections. infections. Therefore,Therefore, it it has has become become a standard a standard small-animal small-animal model model to studyto study infectious infectious colitis colitis [8, 9[8,9].]. Cr Cr infection infection resultsresults in various various changes changes to tothe the colon colon of mice, of mice, including including epithelialepithelial cell proliferation,cell proliferation, crypt crypt hyperplasia, hyperplasia, an an uneven uneven apical apical enterocyte enterocyte surface,surface, cryptcrypt dilation, and mucosaland thickeningmucosal thickening [10]. Except [10]. in Except highly in susceptible highly susceptible mouse strains mouse suchstrains as C3Hsuch/ HeN,as C3H/HeN, colonization of Crcolonization is limited to of the Cr colon, is limited with to few the bacteria colon, with reaching few bacteria systemic reaching organs andsystemic the bloodstreamorgans and the [11 ,12]. Followingbloodstream oral administration, [11,12]. Following Cr oral initially administration, colonizes Cr the initially cecal patchcolonizes and the then cecal migrates patch and to thethen colon migrates to the colon by day 3 post-infection. Bacterial load in the distal colon peaks by day 7 and by day 3 post-infection. Bacterial load in the distal colon peaks by day 7 and remains at high levels remains at high levels through day 12, and is typically cleared by day 21 [13]. In general, Cr infection through day 12, and is typically cleared by day 21 [13]. In general, Cr infection can serve as a useful can serve as a useful model for studying compounds that may prevent or mitigate the effects of EPEC modelor forEHEC studying infections. compounds that may prevent or mitigate the effects of EPEC or EHEC infections. Indole-3-carbinolIndole-3-carbinol (I3C) (I3C) is is aa dietarydietary compound compound (Figure (Figure 1)1 )derived derived from from glucobrassicin, glucobrassicin, a a glucosinolateglucosinolate found found inin cruciferous vegetables vegetables cruciferous cruciferous vegetables vegetables such as such broccoli, as broccoli, cabbage and cabbage and cauliflower.cauliflower. The concentration concentration of ofI3C I3C from from cruciferous cruciferous vegetables vegetables can be can inferred be inferred from the from content the contentof of glucobrassicinglucobrassicin which which varies varies inin didifferentfferent kinds kinds of of cruciferous cruciferous vegetables, vegetables, ranging ranging from from 0.24 0.24μmol·g-1µmol g 1 · − DW toDW 6.2 toµ 6.2mol μmol·g-1g 1 DW DW [14 [14,15].,15]. I3C I3C and and itsits derivativesderivatives ha haveve attracted attracted increased increased attention attention due dueto their to their · − importantimportant role role as anti-inflammatory, as anti-inflammatory, anti-tumor anti-tumor andand immune modulating modulating agents agents [16,17]. [16,17 Substantial]. Substantial evidence indicates that the anti-inflammatory and anti-cancer effects of I3C and its metabolic evidence indicates that the anti-inflammatory and anti-cancer effects of I3C and its metabolic derivatives derivatives are attributed to their ability to modulate several nuclear transcriptional factors including are attributed to their ability to modulate several nuclear transcriptional factors including the estrogen the estrogen receptor (ER), nuclear factor-κB (NF-κB), and the aryl hydrocarbon receptor (AhR), receptorwhich (ER), contribute nuclear to factor- maintainingκB (NF- hormonalκB), and homeosta the aryl hydrocarbonsis, inhibiting receptorcell cycle (AhR),progression/apoptosis, which contribute to maintaininginducing hormonal DNA repair, homeostasis, and enhancing inhibiting carcinogen cell metabolism cycle progression [17,18]. Although/apoptosis, the inducing potential value DNA of repair, and enhancingI3C and its carcinogen derivativesmetabolism in cancer prevention [17,18]. Although and therapy the are potential well known, value ofthe I3C exact and underlying its derivatives in cancermechanisms prevention are still and unclear. therapy are well known, the exact underlying mechanisms are still unclear.

Figure 1. The chemical structure and pharmacological activities of indole-3-carbinol (I3C). Figure 1. The chemical structure and pharmacological activities of indole-3-carbinol (I3C).

Infection-inducedInfection-induced inflammation inflammation is is a a common common risk factor factor for for certain certain types types of cancer. of cancer. Cr infection Cr infection inducesinduces both both innate innate and adaptiveand adaptive immunity, immunity, involving involving the the recruitment recruitment of immuneof immune cells cells and and the the release of multiplerelease cytokinesof multiple andcytokines antimicrobial and antimicrobial peptides peptides that, are that, required are required for clearance for clearance of Cr of [ 19Cr– [19–21]. It is well known21]. It is thatwell IL-22known producing that IL-22 CD4producing+ T cells CD4 are+ T essentialcells are essential for controlling for controlling Cr infection; Cr infection; however, the Thowever, cell-derived the T cytokinescell-derived (IL17A, cytokines IFN- (IL17A,γ and IFN- TNF-γ andα) TNF- contributeα) contribute to intestinal to intestinal tissue tissue injury injury either directlyeither or indirectlydirectly or [22indirectly,23]. In [22,23]. addition Into addition CD4+ Tto cells, CD4+ the T cells, group the 3 innategroup 3 lymphoid innate lymphoid cells (ILC3s) cells also produce(ILC3s) IL22, also which produce is crucial IL22, which for clearance is crucial of for Cr clearance [24]. AhR-deficient of Cr [24]. AhR-deficient mice lack IL-22-producing mice lack IL-22- ILC3 producing ILC3 in the intestinal lamina propria, and have increased mortality when infected with 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 Nutrients 2020, 12, 3148 3 of 15 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 [27–29].

2.2. Cr Infection Mice were infected with Cr using established protocols [26,30,31]. 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 Nutrients 2020, 12, 3148 4 of 15 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 (Table1):

Table 1. The list of PCR primers used in this study.

Primers Catalog Number Primers Catalog Number TBP Mm00446971_m1 IL-1β Mm00434228_m1 Cyp1a1 Mm00487217_m1 TNF-α Mm00443258_m1 IL-17A Mm00439618_m1 IFN-γ Mm01168134_m1 IL-22 Mm01226722_g1 Cd8a Mm01182108_m1 IL-6 Mm00446190_m1 Cd8b Mm00438116_m1 Foxp3 Mm00475162_m1 FasL Mm00438864_m1 Cd4 Mm00442754_m1 Cd5 Mm00432417_m1 Cd19 Mm00515420_m1 Cd45 Mm01293577_m1 Itgam Mm00434455_m1 F4/80 Mm00802529_m1 PD-1 Mm00435532_m1 PD-L1 Mm03048248_m1

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 Nutrients 2020, 12, 3148 5 of 15

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 forNutrients 30 min2020, 12 at, x FOR 37 ◦PEERC, followedREVIEW by the addition of TMB substrate (preheated at5 of 3716 ◦C, mix before using) for colorimetric detection. The reaction was stopped by adding 50 µL of 4N H2SO4 Biotinylated anti-mouse IgG/IgM (Vector Laboratories, Burlingame, CA, USA) was added to each and the absorbancewell. After at30 450min nmof incubation was measured at 37 °C, immediately the plates wereusing washed a 4X microtiter with PBS-T plate and reader50 μL/well (Molecular Devices, Sunnyvale,streptavidin-HRP CA, USA). (1:1000 Data dilution is expressed from stock) as (Vector either theLaboratories, OD450 nmBurlingame, or the ratio CA, ofUSA) infected was OD450 nm/uninfectedadded. OD450 The plates nm. 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 2.8. Statistcal50 AnalysisμL 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 Resultsratio are of expressed infected OD450 as the nm/uninfected mean standard OD450 nm. deviation (SD). Statistical analysis of this study was ± conducted by using GraphPad Prism 7 (2018, GraphPad Software, San Diego, CA, USA). Significance of 2.8. Statistcal Analysis differences between the mean of each group were analyzed using Student’s t test or one-way ANOVA Results are expressed as the mean ± standard deviation (SD). Statistical analysis of this study followed by Fisher’s LSD test. In figures where more than two treatment groups are compared, groups was conducted by using GraphPad Prism 7 (2018, GraphPad Software, San Diego, CA, USA). with differentSignificance letter are of statisticallydifferences between significantly the mean of di ffeaerentch group (p valuewere analyzed< 0.05). using Student’s t test or one-way ANOVA followed by Fisher’s LSD test. In figures where more than two treatment groups 3. Results are compared, groups with different letter are statistically significantly different (p value < 0.05).

3.1. Eﬀect of3. Cr Results Infection and I3C Supplementation on Food Intake and Body Weight

Figure3.1.2A Effect illustrates of Cr Infection the temporal and I3C Supple changesmentation in on body Food weightIntake and of Body uninfected Weight and infected C3H /HeN mice fed controlFigure or I3C 2A diet.illustrates In comparison the temporal changes with the in body control weight uninfected of uninfected group, and infected Cr infection C3H/HeN resulted in weight lossmice in mice fed control fed either or I3C diet. diet. In Infected comparison mice with fed the the control control uninfected diet lost group, a significant Cr infection amountresulted of body weight, whilein weight the body loss in weight mice fed of either infected diet. Infected mice on mice the fed I3C the dietcontrol only dietdecreased lost a significant slightly. amount The of weights body weight, while the body weight of infected mice on the I3C diet only decreased slightly. The of infectedweights mice on of infected the control mice on diet the control were significantlydiet were significantly lower lower than than that that of of infectedinfected mice mice on the on the I3C diet at variousI3C diet days’ at various post-infection days’ post-infection (4, 9, 10, (4, 9, 11). 10, 11). Additionally, Additionally, there there was was a significant a significant drop in dropfood in food consumptionconsumption after the after infection the infection (Figure (Figure2B), 2B), but but it it was was recoveredrecovered by by the the end end of the of experiment, the experiment, and and there were nothere significant were no significant differences differences in food in food consumption consumption between the the four four experimental experimental groups. groups.

7 A B 140 6 Ctrl I3C Ctrl I3C Ctrl-Cr I3C-Cr Ctrl-Cr I3C-Cr 5 130 4

120 3 * * * * 2 110

Relative body weight (%) Infection 1 Infection

100 Food consumption (g/mouse/day) 0 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 Time (days) Time (days)

Figure 2. I3CFigure ameliorates 2. I3C ameliorates the loss the of loss body of body weight weight caused caused by by CitrobacterCitrobacter rodentium rodentium (Cr) infection(Cr) infection but but has no effecthas on no foodeffect consumptionon food consumption in mice. in mice. (A ()A Body) Body weight,weight, the the body body weight weight of mice of on mice different on different days was normalizeddays was normalized to each animal’sto each animal’s body body weight weight on on day day 0. 0. ( B(B)) FoodFood consumption, consumption, infection infection with with Cr Cr was initiated on day 15 after start of the diet. Results were expressed as mean or mean +/- SD (n = was initiated on day 15 after start of the diet. Results were expressed as mean or mean +/ SD (n = 8), 8), * indicates significant difference between the Ctrl-Cr and I3C-Cr groups (p < 0.05). Only within-− * indicates significantday comparisons diff erencewere made. between Ctrl: control the Ctrl-Crdiet; Ctrl-C andr: control I3C-Cr diet, groups Cr-infected; (p < I3C:0.05). I3C Onlydiet; I3C- within-day comparisonsCr: wereI3C diet, made. Cr-infected. Ctrl: control diet; Ctrl-Cr: control diet, Cr-infected; I3C: I3C diet; I3C-Cr: I3C diet, Cr-infected.

3.2. Eﬀects of Dietary I3C on Cr Colonization in Feces and Colon Tissue

In both diet groups, mice had significant levels of Cr in the feces by day 4 post-infection that was substantially cleared by day 20 (Figure3A). There were no di fferences in fecal Cr load between the two dietary treatments during the infection. Moreover, there were no significant differences in colon Cr colonization on day 12 post-infection between mice fed the control or I3C diet (Figure3B). Nutrients 2020, 12, x FOR PEER REVIEW 6 of 16

3.2. Effects of Dietary I3C on Cr Colonization in Feces and Colon Tissue In both diet groups, mice had significant levels of Cr in the feces by day 4 post-infection that was substantially cleared by day 20 (Figure 3A). There were no differences in fecal Cr load between the two dietary treatments during the infection. Moreover, there were no significant differences in colon Nutrients 2020, 12, 3148 6 of 15 Cr colonization on day 12 post-infection between mice fed the control or I3C diet (Figure 3B).

14 14 A Ctrl-Cr B 12 I3C-Cr 12 10 10 b b 8 8

6 6 CFU per g colon colon g per CFU CFU per g feces g feces per CFU 10 10 4 4

Log a a Log 2 2

0 0 0 2 4 6 8 1012141618202224 Ctrl Ctrl-Cr I3C I3C-Cr Days after infection

Figure 3. I3CFigure has 3. no I3C impact has no onimpact Cr on burden Cr burden in feces in feces and and colon colon of of Cr-susceptible Cr-susceptible C3H/HeN C3H /mice.HeN Mice mice. Mice 9 were infectedwere orally infected with orally approximately with approximately 2.5 2.510 × 10CFU9 CFU ofof Cr. (A (A): ):Fecal Fecal excretion excretion of Cr. ofThe Cr. fecal The fecal × pellets of micepellets were of mice collected were collected on di ffonerent different days days after after infection infection and and the the Cr load Crload in feces in (cfu/g feces feces) (cfu /g feces) were determined.were determined. (B): Colonic (B): Colonic colonization colonization of Cr.of Cr. Mice Mice werewere sacrificed sacrificed on onday day12-post-infection 12-post-infection and and the amount of colon tissue associated Cr was determined. Results were expressed as mean +/− SD (n the amount of colon tissue associated Cr was determined. Results were expressed as mean +/ SD = 8). Ctrl: control diet; Ctrl-Cr: control diet, Cr-infected; I3C: I3C diet; I3C-Cr: I3C diet, Cr-infected. − (n = 8). Ctrl:The control dashed diet; line indicates Ctrl-Cr: the control limit of diet,detect Cr-infected;ion of the assay. I3C: Significant I3C diet; differences I3C-Cr: (p < I3C 0.05) diet, between Cr-infected. The dashedgroups line indicates are identified the by limit different of detection letters. of the assay. Significant differences (p < 0.05) between groups are identified by different letters. 3.3. Effects of Dietary I3C on Colon and Cecum Weight in Mice 3.3. Effects of DietaryAfter oral I3C gavage on Colon with andCr, mice Cecum develop Weight colitis, in leading Mice to a thickened and often shortened colon, and less well-formed stools. As shown in Figure S1, the weight of colon and cecum were significantly After oralincreased gavage in infected with Cr, mice mice compared develop to colitis,uninfected leading mice on to both a thickened days 12 and and 21 often post-infection. shortened colon, and less well-formedHowever, there stools. was no As effect shown of I3C in on Figure colon an S1,d cecum the weight weights of when colon comparing and cecum infected were groups. significantly increased in infected mice compared to uninfected mice on both days 12 and 21 post-infection. However, 3.4. Effects of Dietary I3C on Histologic Changes in Colon of Mice there was no effect of I3C on colon and cecum weights when comparing infected groups. Colon tissues obtained on day 12 and day 21 post-infection were processed for H&E staining 3.4. Effects of(Figure Dietary S2). I3C The ondegree Histologic of colon Changes pathology in in Colon mice on of each Mice diet was determined by assessing colon sections. Cr infection resulted in colonic mucosal damage, including increased mucosal thickness, Colonloss tissues of crypt obtained architecture, onday crypt 12 abscesses, and day and 21 eros post-infectionions. Specifically, were infected processed mice fed the for I3C H&E diet staining (Figure S2).had The significantly degree of lower colon mucosal pathology thickness in on mice day 12 on post-infection each diet wascompared determined to the infected by assessingmice fed colon the control diet (Figure 4). On day 21, when the Cr infection was essentially cleared, mucosal sections. Cr infection resulted in colonic mucosal damage, including increased mucosal thickness, thickness in infected mice was reduced compared to day 12. However, there was no significant loss of cryptdifferences architecture, in the crypt mucosal abscesses, thickness between and erosions. infected Specifically,mice fed control infected and I3C micediet on fed day the 21 after I3C diet had significantlyinfection lower (Figure mucosal 4). thicknessIn general, there on day appeared 12 post-infection to be little differences compared in pathology to the of infected colon tissues mice fed the control dietbetween (Figure the4). diet On groups. day 21, when the Cr infection was essentially cleared, mucosal thickness in infected mice was reduced compared to day 12. However, there was no significant differences in the mucosal thickness between infected mice fed control and I3C diet on day 21 after infection (Figure4). In general, thereNutrients appeared 2020, 12, x FOR to PEER be littleREVIEW di fferences in pathology of colon tissues between the7 of diet 16 groups.

500 500 Day 12 Day 21

400 b 400 c 300 300 b b

200 a a 200 a a

100 100 Mucosal thickness (µm) thickness Mucosal Mucosal thickness (µm)Mucosal thickness

0 0 Ctrl Ctrl-Cr I3C I3C-Cr Ctrl Ctrl-Cr I3C I3C-Cr

Figure 4. I3CFigure reduces 4. I3C mucosalreduces mucosal thickness thickness at dayat day 12 12 but but notnot day day 21 21 post-infection. post-infection. Colon Colontissue was tissue was collected oncollected day 12 on and day 12 day and 21 day after 21 after infection infection and processed processed for H&E-staining. for H&E-staining. Mucosal thickness Mucosal was thickness was measuredmeasured on well-oriented on well-oriented crypts crypts and and 12 12 or ormore more individual individual measurements measurements were averaged were foraveraged each for each mouse.mouse. Results Results were were expressed expressed asas mean +/+/− SD SD(n = (n 8).= Ctrl:8). control Ctrl: controldiet; Ctrl- diet;Cr: control Ctrl-Cr: diet,control Cr- diet, infected; I3C: I3C diet; I3C-Cr: I3C diet, Cr-infected.− Significant differences (p < 0.05) between groups Cr-infected;are I3C: identified I3C diet; by different I3C-Cr: letters. I3C diet, Cr-infected. Significant differences (p < 0.05) between groups are identified by different letters. 3.5. Effects of Dietary I3C on AhR and Immune Markers in Colonic Tissues Molecular changes related to AhR and immune pathways were analyzed to help elucidate potential mechanisms of action for I3C (Figure 5). Intake of 1 μmol I3C/g diet significantly induced the expression of the AhR-responsive gene cytochrome P450 1A1 (Cyp1a1) in colonic tissue as compared to animals on control diet. Cr infection significantly attenuated I3C-induced expression of Cyp1a1 mRNA. As expected, mice infected with Cr had increased colonic tissue expression of several cytokines associated with a pro-inflammatory Th1/Th17 immune response including IL-17A, IL-22, IL-6, IL-1β, TNF-α, and IFN-γ on day 12 after infection. Cr-induced expression of IL-17A, IL-6, IL-1β, TNF-α, and IFN-γ mRNA were significantly attenuated in infected mice fed the I3C diet. In contrast, I3C did not affect Cr-induced increase of IL-22 mRNA as compared to control diet. In addition, we also determined gene expression of several markers for CD8+ cell including cd8a, cd8b and FasL. As shown in Figure S3, these markers were upregulated by Cr infection but were not affected by dietary I3C.

Nutrients 2020, 12, 3148 7 of 15

3.5. Effects of Dietary I3C on AhR and Immune Markers in Colonic Tissues Molecular changes related to AhR and immune pathways were analyzed to help elucidate potential mechanisms of action for I3C (Figure5). Intake of 1 µmol I3C/g diet significantly induced the expression of the AhR-responsive gene cytochrome P450 1A1 (Cyp1a1) in colonic tissue as compared to animals on control diet. Cr infection significantly attenuated I3C-induced expression of Cyp1a1 mRNA. As expected, mice infected with Cr had increased colonic tissue expression of several cytokines associated with a pro-inflammatory Th1/Th17 immune response including IL-17A, IL-22, IL-6, IL-1β, TNF-α, and IFN-γ on day 12 after infection. Cr-induced expression of IL-17A, IL-6, IL-1β, TNF-α, and IFN-γ mRNA were significantly attenuated in infected mice fed the I3C diet. In contrast, I3C did not affect Cr-induced increase of IL-22 mRNA as compared to control diet. In addition, we also determined gene expression of several markers for CD8+ cell including cd8a, cd8b and FasL. As shown inNutrients Figure 2020 S3,, these 12, x FOR markers PEER REVIEW were upregulated by Cr infection but were not affected by dietary I3C.8 of 16

500 4000 150 Cyp1a1 IL-17A IL-22 400 3000 b 300 2000 100 b b 200 c b 1000 100 50 20 c 5 5 15 4 4 a 3 a a 3 a a 10 2 2 a 5 1 1 Relative expressionmRNA level Relativeexpression mRNA level Relativeexpression mRNA level 0 0 0 Ctrl Ctrl-Cr I3C I3C-Cr Ctrl Ctrl-Cr I3C I3C-Cr Ctrl Ctrl-Cr I3C I3C-Cr

60 30 30 IL-1β IL-6 TNF-α 50 b 25 25 b

40 20 20 b

30 15 15 c c 20 c 10 10

10 5 5 a a a a a a 0 0 0 Relative mRNA expression level mRNA Relative Relative mRNAexpression level Relative mRNA expression level mRNA Relative Ctrl Ctrl-Cr I3C I3C-Cr Ctrl Ctrl-Cr I3C I3C-Cr Ctrl Ctrl-Cr I3C I3C-Cr

200 25 8 IFN-γ PD-1 PD-L1 150 b 20 100 6 b c b 50 15 c 4 2.0 a c 10 1.5 a 1.0 2 5 a a 0.5 a a RelativemRNAexpression level 0.0 0 Relative mRNA expressionlevel Relative mRNA expression level mRNA Relative 0 Ctrl Ctrl-Cr I3C I3C-Cr Ctrl Ctrl-Cr I3C I3C-Cr Ctrl Ctrl-Cr I3C I3C-Cr

Figure 5. I3C significantly increased xenobiotic metabolizing enzyme genes (Cyp1a1), and attenuated Figure 5. I3C significantly increased xenobiotic metabolizing enzyme genes (Cyp1a1), and attenuated inflammatory markers (IL17A, IL6, IL-1β, TNF-α and IFN-γ) and immune checkpoint inhibitors (PD-1 inflammatory markers (IL17A, IL6, IL-1β, TNF-α and IFN-γ) and immune checkpoint inhibitors (PD- and PD-L1) gene expression in the colon of infected mice, while it has no effect on Cr-induced increase 1 and PD-L1) gene expression in the colon of infected mice, while it has no effect on Cr-induced of IL-22 mRNA. RT-PCR was performed on total RNA isolated from colon tissues harvested on day 12 increase of IL-22 mRNA. RT-PCR was performed on total RNA isolated from colon tissues harvested post-infection. Results are expressed as the mean +/ SD fold-change (n = 8). Ctrl: control diet; Ctrl-Cr: on day 12 post-infection. Results are expressed as− the mean +/− SD fold-change (n = 8). Ctrl: control control diet, Cr-infected; I3C: I3C diet; I3C-Cr: I3C diet, Cr-infected. Significant differences (p < 0.05) diet; Ctrl-Cr: control diet, Cr-infected; I3C: I3C diet; I3C-Cr: I3C diet, Cr-infected. Significant between groups are identified by different letters. differences (p < 0.05) between groups are identified by different letters.

3.6. Effects of Dietary I3C on Cytokine Levels in Serum Circulating cytokines levels in mouse serum were also assessed. Cr infection triggered an increase in serum cytokines in Cr-infected mice fed the control diet, especially increasing the levels of IL17, TNF-α, IL12 (p40, p70) and G-CSF (Figure 6). Overall, the levels of serum cytokines in uninfected mice were not affected by diet. More importantly, in comparison to Cr-infected mice fed the control diet, significant reductions in serum levels of IL17, TNF-α, IL12 (p40, p70) and G-CSF were observed in the Cr-infected mice fed the I3C diet, decreasing them to levels near those observed in uninfected mice fed either diet.

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3.6. Effects of Dietary I3C on Cytokine Levels in Serum Circulating cytokines levels in mouse serum were also assessed. Cr infection triggered an increase in serum cytokines in Cr-infected mice fed the control diet, especially increasing the levels of IL17, TNF-α, IL12 (p40, p70) and G-CSF (Figure6). Overall, the levels of serum cytokines in uninfected mice were not affected by diet. More importantly, in comparison to Cr-infected mice fed the control diet, significant reductions in serum levels of IL17, TNF-α, IL12 (p40, p70) and G-CSF were observed in the

Cr-infectedNutrients 2020 mice, 12, x fed FOR the PEER I3C REVIEW diet, decreasing them to levels near those observed in uninfected mice9 fedof 16 either diet.

7000 800 100 IL-17 TNF-α IL-12 (p40) 6000 700 80 600 5000Nutrients 2020, 12, x FOR PEER REVIEW 9 of 16 500 b 4000 b 60 b 7000 400 800 100 3000 IL-17 TNF-α IL-12 (p40) 6000 300 700 40 a 80 a a 2000 a 600 5000 200 c Concentration (pg/ml) Concentration (pg/ml) Concentration Concentration (pg/ml) 20 a a 500 a b a 60 1000 4000 b 100 b 400 3000 0 0 300 40 0 Ctrl Ctrl-CR I3C I3C-CR Ctrl Ctrl-CR I3C I3C-CRa a Ctrl Ctrl-CRa I3C I3C-CR 2000 a 200 c Concentration (pg/ml) Concentration (pg/ml) Concentration Concentration (pg/ml) a 20 1000 a a a 1200 100 500 0 IL-12 (p70) 0 G-CSF 0 Ctrl Ctrl-CR I3C I3C-CR Ctrl Ctrl-CR I3C I3C-CR Ctrl Ctrl-CR I3C I3C-CR 1000 400 1200 500 IL-12 (p70) G-CSF 800 b 1000 300 400 b 600 800 b 300 200 b 400 600 a 200 a a 400 Concentration (pg/ml) 100

Concentration (pg/ml) Concentration 200 a a a a a Concentration (pg/ml) 100

Concentration (pg/ml) Concentration 200 a a a 0 a 0 Ctrl0 Ctrl-CR I3C I3C-CR 0 Ctrl Ctrl-CR I3C I3C-CR Ctrl Ctrl-CR I3C I3C-CR Ctrl Ctrl-CR I3C I3C-CR

Figure 6. I3C significantly decreased Cr-induced levels of IL17, TNF-α, IL-12, and G-CSF in serum of Figure 6. I3CFigure significantly 6. I3C significantly decreased decreased Cr-induced Cr-induced levels levels ofof IL17, IL17, TNF- TNF-α, IL-12,α, IL-12, and G-CSF and inG-CSF serum inof serum of infected mice fed control diet. Results are expressed as the mean +/ SD (n = 8). Ctrl: control diet; infected miceinfected fed micecontrol fed control diet. Results diet. Results are are expressed expressed asas thethe mean mean +/− +/ SD− (n−SD = 8).(n Ctrl: = 8). control Ctrl: diet;control Ctrl- diet; Ctrl- Ctrl-Cr: controlCr: control diet, diet, Cr-infected; Cr-infected; I3C: I3C: I3CI3C diet; diet; I3C-Cr I3C-Cr:: I3C I3Cdiet, diet,Cr-infected. Cr-infected. Significant Significant differences ( dip <ff erences Cr: control diet, Cr-infected; I3C: I3C diet; I3C-Cr: I3C diet, Cr-infected. Significant differences (p < (p < 0.05) between0.05) between groups groups are are identified identifiedby by different different letters. letters. 0.05) between groups are identified by different letters. 3.7. Effects of3.7. Dietary Effects of I3C Dietary on Spleen I3C on Spleen Size andSize Immuneand Immune Cell Cell Molecular Molecular Markers Markers 3.7. Effects of DietaryWe found I3C Cr on infection Spleen Sizeled to and a significantImmune Cellincrease Molecular in spleen Markers size only in the Cr sensitive We found Cr infection led to a significant increase in spleen size only in the Cr sensitive C3H/HeN C3H/HeN mice, while Cr infection did not affect the spleen size of Cr-resistant C57BL/J6 mice (Figure We found Cr infection led to a significant increase in spleen size only in the Cr sensitive mice, while7). Cr Consumption infection of did dietary not I3C aff ledect to the a significant spleen attenuation size of Cr-resistant of the Cr-induced C57BL increase/J6 micein spleen (Figure 7). ConsumptionC3H/HeN sizemice, of indietary C3H/HeN while Cr I3C mice. infection led The to effect a significantdid of not Cr and affect I3C attenuation onthe sp spleenleen size of size thewas still Cr-inducedof Cr-resistant observed even increase C57BL/J6at 21 days in spleen post- mice size(Figure in C3H7). Consumption/HeN mice.infection. The of e ffdietaryect of CrI3C and led I3C to ona significant spleen size attenuation was still observed of the evenCr-induced at 21 days increase post-infection. in spleen size in C3H/HeN mice. The effect of Cr and I3C on spleen size was still observed even at 21 days post- infection.

Figure 7. CrFigure infection 7. Cr infection resulted resulted in a significant in a significant increase increase in in spleenspleen size size of ofC3H/HeN C3H/HeN mice while mice having while having no effect onno the effect spleen on the size spleen of C57BLsize of C57BL/J6/J6 mice. mice. I3C I3C significantly significantly attenuated attenuated Cr-induced Cr-induced increase increase in in spleen size inspleen C3H size/HeN in C3H/HeN mice. Ctrl: mice. control Ctrl: control diet; diet; Ctrl-Cr: Ctrl-Cr: control control diet,diet, Cr-infec Cr-infected;ted; I3C: I3C: I3C I3Cdiet; diet;I3C- I3C-Cr: I3C diet, Cr-infected.Cr: I3C diet, Significant Cr-infected. diSignificantfferences differences (p < 0.05) (p between< 0.05) between groups groups are are identified identified by by didifferentfferent letters. letters.

Figure 7. Cr infection resulted in a significant increase in spleen size of C3H/HeN mice while having no effect on the spleen size of C57BL/J6 mice. I3C significantly attenuated Cr-induced increase in

spleen size in C3H/HeN mice. Ctrl: control diet; Ctrl-Cr: control diet, Cr-infected; I3C: I3C diet; I3C- Cr: I3C diet, Cr-infected. Significant differences (p < 0.05) between groups are identified by different letters.

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TheThe molecular molecular e ffeffectsects of of spleen spleen enlargement enlargement were were further further assessed assessed using using specific specific markers markers for for immuneimmune cells cells (Figure (Figure8). 8). We We observed observed an increasean increase in macrophage in macrophage makers makers such such as Itgam as Itgam and F4and/80 F4/80 in the in Cr-infectedthe Cr-infected mice. Themice. induction The induction of macrophage of macrophage markers bymarkers Cr-infection by Cr-infection was significantly was significantly attenuated inattenuated the I3C-fed in group.the I3C-fed Consistent group. with Consistent cytokine with expression cytokine inexpressi the colon,on in proinflammatory the colon, proinflammatory cytokines suchcytokines as IL-1 suchβ and as IL-6IL-1β were and inducedIL-6 were by induced Cr and by attenuated Cr and attenuated by dietary by I3C dietary in spleens I3C in of spleens Cr-infected of Cr- mice.infected However, mice. Cr-infectionHowever, Cr-infection significantly significantly decreased expression decreased of B-expression and T-cell of markers B- and (CD19, T-cell CD45R;markers CD4,(CD19, CD8a) CD45R; in the CD4, spleen. CD8a) in the spleen.

FigureFigure 8. 8.I3C I3C significantly significantly attenuated attenuated Cr-induced Cr-induced increases increases in in mRNA mRNA levels levels of of macrophage macrophage makers makers (Itgam and F4/80) and proinflammatory cytokines (IL-1β and IL-6) in the spleen. Results are expressed (Itgam and F4/80) and proinflammatory cytokines (IL-1β and IL-6) in the spleen. Results are expressed as the mean +/ SD fold-change (n = 8). Ctrl: control diet; Ctrl-Cr: control diet, Cr-infected; I3C: as the mean +/−− SD fold-change (n = 8). Ctrl: control diet; Ctrl-Cr: control diet, Cr-infected; I3C: I3C I3C diet; I3C-Cr: I3C diet, Cr-infected. Significant differences (p < 0.05) between groups are identified diet; I3C-Cr: I3C diet, Cr-infected. Significant differences (p < 0.05) between groups are identified by by different letters. different letters. 3.8. Effects of Dietary I3C on Cr-Specific Antibody Levels in Serum 3.8. Effects of Dietary I3C on Cr-Specific Antibody Levels in Serum To further investigate potential mechanisms by which I3C elicits protective effects, we also determinedTo further the influenceinvestigate of potential I3C on mechanisms Cr-specific serum by which antibody I3C elicits production protective by usingeffects, ELISAs.we also Considerabledetermined variationsthe influence in antigen-specific of I3C on Cr-specific IgA, IgG, andserum IgM antibody production production were noted by among using C57BL ELISAs./J6 andConsiderable C3H/HeN micevariations in each in dietary antigen-specific group on day IgA, 12 afterIgG, infection.and IgM Theproduction data when were expressed noted asamong OD valuesC57BL/J6 (Figure and S4) C3H/HeN indicates mice that I3Cin attenuatedeach dietary the grou antibodyp on responseday 12 after in resistant infection. C57BL The/J6 data mice when but notexpressed in susceptible as OD C3Hvalues/HeN (Figure mice S4) but indicates similarly that reduced I3C attenuated the IgM response the antibody in the response two strains. in resistant Serum IgAC57BL/J6 responses mice were but lowernot in and susceptible not affected C3H/HeN by diet mice in either but strain.similarly However, reduced it the was IgM consistently response noted in the thattwo the strains. background Serum IgA levels responses of reactivity were werelower lower and not in I3C affected treated by mice.diet in The either reason strain. for However, this is not it clear.was consistently But when the noted data that was the expressed background as fold levels induction of reactivity over backgroundwere lower in dietary I3C treated I3C treatment mice. The ledreason to an for enhanced this is not Cr-specific clear. But IgG when response the data in Cr-infected was expressed C3N/HeN as fold mice induction but not inover C57BL background/J6 mice (Figuredietary9). I3C The treatment e ffects of led Cr to infection an enhanced on Cr-specific Cr-specific IgM IgG and response IgA were in similarCr-infected in control- C3N/HeN and I3C-fedmice but mice.not in In C57BL/J6 contrast, themice IgG (Figure and IgA 9). The responses effects to of Cr Cr infection infection were on Cr-specific similar in Cr-resistantIgM and IgA C57BL were/6 similar mice onincontrol control- and and I3C I3C-fed diet, while mice. I3C-fed In contrast, mice’s the IgM IgG responses and IgA was responses significantly to Cr attenuated,infection were as compared similar in withCr-resistant that of infected C57BL/6 control mice group. on control and I3C diet, while I3C-fed mice’s IgM responses was significantly attenuated, as compared with that of infected control group.

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Sensitive mice Resistant mice (C3H/HeN) (C57BL/J6)

120 20 d 100 15 b 80 b

60 10 c 40 5 aa

IgG Fold Induction Fold IgG 20 Induction Fold IgG a a 0 0 Ctrl Ctrl-Cr I3C I3C-Cr Ctrl Ctrl-Cr I3C I3C-Cr Treatments Treatments

30 50 c b 25 40 b 20 30 15 20 c 10

10 IgM Fold Induction Fold IgM 5 a a Induction Fold IgM a a 0 0 Ctrl Ctrl-Cr I3C I3C-Cr Ctrl Ctrl-Cr I3C I3C-Cr Treatments Treatments

30 80

25 c 60 b b 20

15 b 40

10 20

IgA Fold Induction Fold IgA 5 a Induction Fold IgA a a a 0 0 Ctrl Ctrl-Cr I3C I3C-Cr Ctrl Ctrl-Cr I3C I3C-Cr Treatments Treatments

Figure 9. Effects of I3C on Cr-induced IgG response in sensitive (C3H/HeN) and resistant (C57BL/J6) Figure 9. Effects of I3C on Cr-induced IgG response in sensitive (C3H/HeN) and resistant (C57BL/J6) mice. Cr-specific serum antibody levels were calculated as fold change between uninfected controls and mice.infected Cr-specific mice on serum each diet. antibody Results levels expressed were as calculated the mean +/ asSD fold fold-change change between (n = 8). Ctrl: uninfected control diet; controls − andCtrl-Cr: infected control mice diet,on each Cr-infected; diet. Results I3C: I3C expressed diet; I3C-Cr: as the I3C mean+/-SD diet, Cr-infected. fold-change Significant (n = di8).ff erencesCtrl: control diet;(p

Nutrients 2020, 12, 3148 11 of 15

Mice infected with Cr often develop acute colitis accompanied by an overgrowth of Cr and self-limiting inflammation in murine intestinal lumen [10,35]. 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 [19,22]. 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 [22,24,37]. 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 [23,38]. Many types of immune cell, such as CD4+ T cell and ILC3 cell, secrete the anti-inflammatory cytokine IL-22 [24,39]. Production of IL-1β and IL-17A and the AhR receptor pathway have been reported to modulate the production of IL-22 [40–42]. 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 Nutrients 2020, 12, 3148 12 of 15

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.

5. Conclusions In summary, the data presented here indicate that the cruciferous vegetable-derived compound I3C can provide an overall anti-inflammatory effect on the host. I3C acts by protecting the host against Cr-infection by maintaining IL-22 levels and reducing expression of other pro-inflammatory cytokines, likely through an AhR-dependent pathway. A protective effect on spleen enlargement and attenuated systematic immune response were also observed and may be related to enhanced IgG response in I3C-fed mice. Hence, it is possible that the host response to E. coli infection may be modified by consumption of I3C or cruciferous vegetables.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/10/3148/s1, Figure S1. I3C has no impact on increased colon and cecum weight of infected mice. The colon and cecum weight of uninfected and Cr-infected mice fed control and I3C diet were measured on day 12 (A) and 21 (B) post-infection. Results were expressed as mean +/ SD (n = 8). Significant differences (p < 0.05) between groups are identified by different letters. Figure S2. Representative− images of H&E stained colon sections of uninfected and Cr-infected mice fed control or I3C diet. The colon tissues were harvested on day 12 (a, b, c, d) and day 21(e, f, g, h) post-infection. (a, e): Uninfected mice fed with control diet; (b, f): Infected mice fed with control diet; (c, g): Uninfected mice fed with I3C diet; (d, h): Infected mice fed with I3C diet. The Cr- infected mice fed control diet had crypt hyperplasia, loss of crypt architecture, crypt abscesses and erosions (panel b). I3C, to a certain extent, could ameliorated the colonic damage caused by the infection on day 12. Original magnification 10. Figure S3. Cr infection significantly increased mRNA levels of CD8+ cell markers (Cd8a, Cd8b and FasL), while× I3C has no effect on gene expression of these markers in infected mice. RT-PCR was performed on total RNA isolated from colon tissues harvested on day 12 post-infection. Results are expressed as the mean +/ SD fold-change (n = 8). Different letters indicated significant differences between groups for each tissue (p < 0.05).− Figure S4. Effects of I3C on Cr-triggered IgG response in Sensitive (C3H/HeN mice) and Resistant (C57BL/J6 mice). Author Contributions: T.T.Y.W., A.D.S. designed the experiment; Y.W., A.D.S., L.C. and Z.Z. performed the animal experiment; Y.W. performed the data analysis; Y.W., J.W., Q.P., Q.H., L.Y., Y.S.K. and T.T.Y.W. participated in the writing, review, and/or revision of the manuscript; T.T.Y.W. and A.D.S. supervised this study. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by U.S. Department of Agriculture appropriated fund #8040-51530-057-00D, non-funded cooperative agreement between USDA and Sichuan University, foundation for Young Scientist of Beijing Technology & Business University (PXM2020_014213_000017, Grant QNJJ2020-11). This work was also supported by an interagency agreement between USDA and the National Cancer Institute, Nutrition Research Group. Acknowledgments: The authors thank Harold Seifried, National Cancer Institute, Nutrition Research Group, fo critically reading of the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Nutrients 2020, 12, 3148 13 of 15

References

1. Petty, N.K.; Bulgin, R.; Crepin, V.F.; Cerdeño-Tárraga, A.M.; Schroeder, G.N.; Quail, M.A.; Lennard, N.; Corton, C.; Barron, A.; Clark, L. The Citrobacter rodentium genome sequence reveals convergent evolution with human pathogenic Escherichia coli. J. Bacteriol. 2010, 192, 525–538. [CrossRef][PubMed] 2. Hartland, E.L.; Leong, J. Enteropathogenic and enterohemorrhagic E. coli: Ecology, pathogenesis, and evolution. Front. Cell. Infect. Microbiol. 2013, 3, 15. [CrossRef][PubMed] 3. Chen, H.D.; Frankel, G. Enteropathogenic Escherichia coli: Unravelling pathogenesis. FEMS Microbiol. Rev. 2005, 29, 83–98. [CrossRef][PubMed] 4. Nataro, J.P.; Kaper, J.B. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 1998, 11, 142–201. [CrossRef] [PubMed] 5. Ryan, C.A.; Tauxe, R.V.; Hosek, G.W.; Wells, J.G.; Stoesz, P.A.; McFadden, H.W., Jr.; Smith, P.W.; Wright, G.F.; Blake, P.A. Escherichia coli O157: H7 diarrhea in a nursing home: Clinical, epidemiological, and pathological findings. J. Infect. Dis. 1986, 154, 631–638. [CrossRef][PubMed] 6. MacDonald, I.A.R.; Gould, I.M.; Curnow, J. Epidemiology of infection due to Escherichia coli O157: A 3-year prospective study. Epidemiol. Infect. 1996, 116, 279–284. [CrossRef][PubMed] 7. Petty, N.K.; Zakour, N.L.B.; Stanton-Cook, M.; Skippington, E.; Totsika, M.; Forde, B.M.; Phan, M.-D.; Moriel, D.G.; Peters, K.M.; Davies, M. Global dissemination of a multidrug resistant Escherichia coli clone. Proc. Natl. Acad. Sci. USA 2014, 111, 5694–5699. [CrossRef] 8. Chandrakesan, P.; Roy, B.; Jakkula, L.; Ahmed, I.; Ramamoorthy, P.; Tawfik, O.; Papineni, R.; Houchen, C.; Anant, S.; Umar, S. Utility of a bacterial infection model to study epithelial–mesenchymal transition, mesenchymal–epithelial transition or tumorigenesis. Oncogene 2014, 33, 2639–2654. [CrossRef] 9. Higgins, L.M.; Frankel, G.; Douce, G.; Dougan, G.; MacDonald, T.T. Citrobacter rodentium infection in mice elicits a mucosal Th1 cytokine response and lesions similar to those in murine inflammatory bowel disease. Infect. Immun. 1999, 67, 3031–3039. [CrossRef] 10. Mundy, R.; MacDonald, T.T.; Dougan, G.; Frankel, G.; Wiles, S. Citrobacter rodentium of mice and man. Cell. Microbiol. 2005, 7, 1697–1706. [CrossRef] 11. Papapietro, O.; Teatero, S.; Thanabalasuriar, A.; Yuki, K.E.; Diez, E.; Zhu, L.; Kang, E.; Dhillon, S.; Muise, A.M.; Durocher, Y. R-spondin 2 signalling mediates susceptibility to fatal infectious diarrhoea. Nat. Commun. 2013, 4, 1–9. [CrossRef][PubMed] 12. Borenshtein, D.; McBee, M.E.; Schauer, D.B. Utility of the Citrobacter rodentium infection model in laboratory mice. Curr. Opin. Gastroenterol. 2008, 24, 32–37. [CrossRef][PubMed] 13. Wiles, S.; Clare, S.; Harker, J.; Huett, A.; Young, D.; Dougan, G.; Frankel, G. Organ specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium. Cell. Microbiol. 2004, 6, 963–972. [CrossRef][PubMed] 14. Kushad, M.M.; Brown, A.F.; Kurilich, A.C.; Juvik, J.A.; Klein, B.P.; Wallig, M.A.; Jeffery, E.H. Variation of Glucosinolates in Vegetable Crops of Brassica o leracea. J. Agric. Food Chem. 1999, 47, 1541–1548. [CrossRef] 15. Padilla, G.; Cartea, M.E.; Velasco, P.; de Haro, A.; Ordás, A. Variation of glucosinolates in vegetable crops of Brassica rapa. Phytochemistry 2007, 68, 536–545. [CrossRef] 16. Keck, A.-S.; Finley, J.W. Cruciferous vegetables: Cancer protective mechanisms of glucosinolate hydrolysis products and selenium. Integr. Cancer Ther. 2004, 3, 5–12. [CrossRef] 17. Aggarwal, B.B.; Ichikawa, H. Molecular targets and anticancer potential of indole-3-carbinol and its derivatives. Cell Cycle 2005, 4, 1201–1215. [CrossRef] 18. Kim, Y.S.; Milner, J.A. Targets for indole-3-carbinol in cancer prevention. J. Nutr. Biochem. 2005, 16, 65–73. [CrossRef] 19. Vallance, B.A.; Deng, W.; Knodler, L.A.; Finlay, B.B. Mice lacking T and B lymphocytes develop transient colitis and crypt hyperplasia yet suffer impaired bacterial clearance during Citrobacter rodentium infection. Infect. Immun. 2002, 70, 2070–2081. [CrossRef] 20. Hall, L.J.; Murphy, C.T.; Hurley, G.; Quinlan, A.; Shanahan, F.; Nally, K.; Melgar, S. Natural killer cells protect against mucosal and systemic infection with the enteric pathogen Citrobacter rodentium. Infect. Immun. 2013, 81, 460–469. [CrossRef] Nutrients 2020, 12, 3148 14 of 15

21. Schreiber, H.A.; Loschko, J.; Karssemeijer, R.A.; Escolano, A.; Meredith, M.M.; Mucida, D.; Guermonprez, P.; Nussenzweig, M.C. Intestinal monocytes and macrophages are required for T cell polarization in response to Citrobacter rodentium. J. Exp. Med. 2013, 210, 2025–2039. [CrossRef] 22. Simmons, C.P.; Clare, S.; Ghaem-Maghami, M.; Uren, T.K.; Rankin, J.; Huett, A.; Goldin, R.; Lewis, D.J.; MacDonald, T.T.; Strugnell, R.A. Central role for B lymphocytes and CD4+ T cells in immunity to infection by the attaching and effacing pathogen Citrobacter rodentium. Infect. Immun. 2003, 71, 5077–5086. [CrossRef] 23. Ota, N.; Wong, K.; Valdez, P.A.; Zheng, Y.; Crellin, N.K.; Diehl, L.; Ouyang, W. IL-22 bridges the lymphotoxin pathway with the maintenance of colonic lymphoid structures during infection with Citrobacter rodentium. Nat. Immunol. 2011, 12, 941. [CrossRef][PubMed] 24. Geddes, K.; Rubino, S.J.; Magalhaes, J.G.; Streutker, C.; Le Bourhis, L.; Cho, J.H.; Robertson, S.J.; Kim, C.J.; Kaul, R.; Philpott, D.J. Identification of an innate T helper type 17 response to intestinal bacterial pathogens. Nat. Med. 2011, 17, 837–844. [CrossRef] 25. Kiss, E.A.; Vonarbourg, C.; Kopfmann, S.; Hobeika, E.; Finke, D.; Esser, C.; Diefenbach, A. Natural aryl hydrocarbon receptor ligands control organogenesis of intestinal lymphoid follicles. Science 2011, 334, 1561–1565. [CrossRef] 26. Wu, Y.; He, Q.; Yu, L.; Pham, Q.; Cheung, L.; Kim, Y.S.; Wang, T.T.Y.; Smith, A.D. Indole-3-Carbinol Inhibits Citrobacter rodentium Infection through Multiple Pathways Including Reduction of Bacterial Adhesion and Enhancement of Cytotoxic T Cell Activity. Nutrients 2020, 12, 917. [CrossRef] 27. Kassie, F.; Matise, I.; Negia, M.; Upadhyaya, P.; Hecht, S.S. Dose-dependent inhibition of tobacco smoke carcinogen–induced lung tumorigenesis in A/J mice by indole-3-carbinol. Cancer Prev. Res. 2008, 1, 568–576. [CrossRef] 28. Fletcher, A.; Huang, H.; Yu, L.; Pham, Q.; Yu, L.; Wang, T.T.Y. Reversible toxic effects of the dietary supplement Indole-3-carbinol in an immune compromised rodent model: Intestine as the main target. J. Diet. Suppl. 2017, 14, 303–322. [CrossRef] 29. Reed, G.A.; Arneson, D.W.; Putnam, W.C.; Smith, H.J.; Gray, J.C.; Sullivan, D.K.; Mayo, M.S.; Crowell, J.A.; Hurwitz, A. Single-dose and multiple-dose administration of indole-3-carbinol to women: Pharmacokinetics

based on 3,30-diindolylmethane. Cancer Epidemiol. Prev. Biomark. 2006, 15, 2477–2481. [CrossRef][PubMed] 30. Smith, A.D.; Cheung, L.; Botero, S. Long-term selenium deficiency increases the pathogenicity of a Citrobacter rodentium infection in mice. Biol. Trace Elem. Res. 2011, 144, 965–982. [CrossRef] 31. Smith, A.; Bhagwat, A.A. Hypervirulent-host-associated Citrobacter rodentium cells have poor acid tolerance. Curr. Microbiol. 2013, 66, 522–526. [CrossRef][PubMed] 32. Cerceo, E.; Deitelzweig, S.B.; Sherman, B.M.; Amin, A.N. Multidrug-resistant gram-negative bacterial infections in the hospital setting: Overview, implications for clinical practice, and emerging treatment options. Microb. Drug Resist. 2016, 22, 412–431. [CrossRef][PubMed] 33. Sung, W.S.; Lee, D.G. In vitro antimicrobial activity and the mode of action of indole-3-carbinol against human pathogenic microorganisms. Biol. Pharm. Bull. 2007, 30, 1865–1869. [CrossRef][PubMed] 34. Bradlow, H.L.; Michnovicz, J.J.; Halper, M.; Miller, D.G.; Wong, G.Y.; Osborne, M.P. Long-term responses of women to indole-3-carbinol or a high fiber diet. Cancer Epidemiol. Prev. Biomark. 1994, 3, 591–595. 35. Barthold, S.W.; Coleman, G.L.; Bhatt, P.N.; Osbaldiston, G.W.; Jonas, A.M. The etiology of transmissible murine colonic hyperplasia. Lab. Anim. Sci. 1976, 26, 889–894. 36. Maaser, C.; Housley, M.P.; Iimura, M.; Smith, J.R.; Vallance, B.A.; Finlay, B.B.; Schreiber, J.R.; Varki, N.M.; Kagnoff, M.F.; Eckmann, L. Clearance of Citrobacter rodentium requires B cells but not secretory immunoglobulin A (IgA) or IgM antibodies. Infect. Immun. 2004, 72, 3315–3324. [CrossRef] 37. Schauer, D.B.; Falkow, S. Attaching and effacing locus of a Citrobacter freundii biotype that causes transmissible murine colonic hyperplasia. Infect. Immun. 1993, 61, 2486–2492. [CrossRef] 38. Liang, S.C.; Tan, X.-Y.; Luxenberg, D.P.; Karim, R.; Dunussi-Joannopoulos, K.; Collins, M.; Fouser, L.A. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J. Exp. Med. 2006, 203, 2271–2279. [CrossRef] 39. Zheng, Y.; Valdez, P.A.; Danilenko, D.M.; Hu, Y.; Sa, S.M.; Gong, Q.; Abbas, A.R.; Modrusan, Z.; Ghilardi, N.; De Sauvage, F.J. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat. Med. 2008, 14, 282–289. [CrossRef] 40. Valeri, M.; Raffatellu, M. Cytokines IL-17 and IL-22 in the host response to infection. Pathog. Dis. 2016, 74, ftw111. [CrossRef] Nutrients 2020, 12, 3148 15 of 15

41. Veldhoen, M.; Hirota, K.; Christensen, J.; O’Garra, A.; Stockinger, B. Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal differentiation of Th17 T cells. J. Exp. Med. 2009, 206, 43–49. [CrossRef][PubMed] 42. Qiu, J.; Guo, X.; Zong-ming, E.C.; He, L.; Sonnenberg, G.F.; Artis, D.; Fu, Y.-X.; Zhou, L. Group 3 innate lymphoid cells inhibit T-cell-mediated intestinal inflammation through aryl hydrocarbon receptor signaling and regulation of microflora. Immunity 2013, 39, 386–399. [CrossRef][PubMed] 43. Busbee, P.B.; Menzel, L.; Alrafas, H.R.; Dopkins, N.; Becker, W.; Miranda, K.; Tang, C.; Chatterjee, S.; Singh, U.P.; Nagarkatti, M. Indole-3-carbinol prevents colitis and associated microbial dysbiosis in an IL-22–dependent manner. JCI Insight 2020, 5, e127551. [CrossRef][PubMed] 44. Bohnsack, J.F.; Brown, E.J. The role of the spleen in resistance to infection. Annu. Rev. Med. 1986, 37, 49–59. [CrossRef][PubMed]

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