Dietary broccoli impacts microbial community structure and attenuates chemically induced colitis in mice in an Ah receptor dependent manner
Introduction
Plants of the genus Brassica, often referred to as cruciferous vegetables (e.g. broccoli, cabbage, Brussels sprouts, etc.), provide critical components of a healthy diet, such as vitamins, minerals, and fiber. Consumption of these plants correlates with decreased incidence of cancer (Latte, Appel, & Lampen, 2011). While individual chemical constituents found in broccoli have been reported in isolation to exhibit beneficial properties, the relative importance of each chemical within the context of dietary broccoli in conferring these beneficial effects is poorly understood (Bradlow et al., 1999, Higdon et al., 2007, Zhang et al., 1992).
Broccoli (Brassica oleracea) has been extensively studied for its content of various nutritional phytochemicals, such as glucosinolates (Kushad et al., 1999). Enzymatic hydrolysis of glucosinolates by plant myrosinases generates numerous metabolites including isothiocyanates, thiocyanates, and epithionitriles (Fenwick, Heaney, & Mullin, 1983) which exhibit diverse biological activities, Perhaps one of the most studied components of this class of glucosides are indole-3-carbinol (I3C), indole-3-acetonitrile (I3ACN) and 3,3′diindolylmethane (DIM), breakdown products of the glucosinolate, glucobrassicin. I3C and its oligomeric products exhibit multiple biological activities, including induction of apoptosis, decreased DNA-adduct formation, and reduced estrogen signaling, but have been characterized and extensively investigated as ligands and modulators of aryl hydrocarbon receptor (AHR) activity (Aggarwal & Ichikawa, 2005). Additionally, gastric acid-mediated condensation of I3C in the stomach yields 2-(indol-3-ylmethyl)-3,3′-diindolylmethane (Ltr-1) and indolo[3,2-b]carbazole (ICZ), a potent ligand and activator of the AHR (Bjeldanes, Kim, Grose, Bartholomew, & Bradfield, 1991). As such, broccoli represents a rich source of dietary AHR ligands (Hubbard, Murray, & Perdew, 2015).
The AHR is a ligand activated transcription factor, of the basic region helix-loop-helix-PER/ARNT/SIM homology super-family, first identified as the mediator of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity (Poland & Knutson, 1982). The receptor possesses a promiscuous ligand binding domain that is responsive to an array of xenobiotic compounds, such as polycyclic aromatic hydrocarbons (PAH’s) and polychlorinated biphenyls (PCBs) (Poland & Knutson, 1982). The AHR has been historically characterized for its capacity to induce metabolism of exogenous compounds through transcriptional regulation of the cytochrome P450 gene battery (Denison et al., 1988, Rowlands and Gustafsson, 1997). The physiological roles of the AHR have expanded beyond xenobiotic metabolism, to include an array of endogenous functions. This concept is supported by the generation and phenotypic characterization of AHR null mice, which exhibit numerous physiological anomalies, including immune dysfunction, decreased hepatic vasculature development, and reduced fertility (Baba et al., 2005, McMillan and Bradfield, 2007).
Additional studies provide evidence that ligand-activated AHR participates in the maintenance of intestinal homeostasis. AHR null mice, relative to their wild-type counterparts, are more susceptible to various modes of intestinal challenge, such as chemically-induced colitis or C. rodentium infection (Li et al., 2011, Qiu et al., 2012). Protection is also conferred in ligand-responsive mice by administration of AHR agonists, such as TCDD and I3C (Furumatsu et al., 2011, Takamura et al., 2010). In the absence of exogenous agonists, AHR activity is likely mediated by ligands produced endogenously or provided via dietary sources, such as cruciferous vegetables. Administration of whole broccoli has been previously shown to alter the resident microflora in mice and to decrease colonic inflammation (Paturi et al., 2012). The mechanism(s) by which broccoli confers these activities and their dependency upon AHR-activation have not been fully investigated.
Here, the impact of broccoli consumption upon AHR activation, intestinal microbial community structure, and intestinal homeostasis is examined in vivo. To delineate the physiological impacts of broccoli consumption that associate with AHR ligand-responsiveness we utilized congenic mice expressing either the Ahrb/b (high-affinity) or Ahrd/d alleles (low-affinity) (Nebert & Bausserman, 1970). Mice harboring the Ahrd/d allele exhibit decreased sensitivity to many prototypical AHR ligands (Poland & Glover, 1975). Data presented here demonstrate that a significant component of the beneficial effects of broccoli can be attributed to activation of the AHR signaling pathway, resulting in an altered microbiome and protection from chemically induced colitis.
Section snippets
Animals and husbandry
All animal studies were performed with approval and under the support of the Institutional Animal Care and Use Committee (IACUC Protocol #45967, The Pennsylvania State University). C57BL6/J mice were originally purchased from Jackson Laboratories (Bar Harbor, ME). C57BL6/J-Ahrb/b and derived C57BL6/J-Ahrd/d were subsequently bred in-house. Animals were housed in autoclaved polypropylene cages with corncob bedding in a specific pathogen-free environment with ad libitum access to indicated diets
Dietary broccoli induces the prototypic AHR target Gene, Cyp1a1
Activation of the AHR in response to dietary administration of whole broccoli was initially assessed in C57BL6/J mice that express the high affinity Ahrb/b allele. Mice were acclimatized to a defined pellet diet of AIN-93G (Low in AHR ligands) for seven days, and then administered finely ground diets containing concentrations of 0% or 10% (w/w) freeze-dried broccoli for an additional seven days. Broccoli at 10% in the diet promoted a significant 3-fold elevation in duodenal Cyp1a1 expression (
Discussion
In the present study, we observed that broccoli mediated a greater level of intestinal AHR activation in Ahrb/b relative to Ahrd/d mice. This is likely an indication of a reduced capacity for the Ahrd/d isoform to bind and respond to broccoli-derived ligands. Previous studies suggest administration of broccoli will activate the AHR; however, these studies fail to consider the array of chemical components present in broccoli that could impact uptake and metabolism of I3C or antagonize the AHR.
Conclusions
Broccoli consumption alters the host microbiome and improves intestinal resistance to chemical challenge, which suggests there is a therapeutic effect on the maintenance of intestinal homeostasis. Most importantly, this latter effect of broccoli consumption is mediated through AHR activation. Therefore, the selection of broccoli cultivars with increased levels of glucosinolates, especially those that lead to the formation of AHR ligands, may be of increased health benefit. Furthermore, numerous
Acknowledgements
We thank the Penn State Genomics Core Facility, the Penn State Metabolomics Core Facility, Animal diagnostic Laboratory, and Gnotobiotic Animal Research Facility- University Park, PA for their many contributions to this project. We also express our gratitude to John Esslinger and Brian Campbell for providing the broccoli cultivar used for the study. We also thank Marcia H. Perdew for excellent editorial assistance.
Funding Sources
This project was supported by Agriculture and Food Research Initiative Competitive
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