Dietary broccoli impacts microbial community structure and attenuates chemically induced colitis in mice in an Ah receptor dependent manner

Highlights

• Dietary broccoli activates the colonic AHR the high ligand affinity AHR^b in mice.

• Dietary broccoli influences the composition of the mouse cecal microbiome.

• Dietary broccoli decreases inflammation in the gastrointestinal tract of AHR^b mice.

• Dietary broccoli protects the GI tract of AHR^b, but not AHR^d mice from toxic insult.

Abstract

Consumption of broccoli mediates numerous chemo-protective benefits through the intake of phytochemicals, some of which modulate aryl hydrocarbon receptor (AHR) activity. Whether AHR activation is a critical aspect of the therapeutic potential of dietary broccoli is not known. Here we administered isocaloric diets, with or without supplementation of whole broccoli (15% w/w), to congenic mice expressing the high-affinity Ahr^b/b or low-affinity Ahr^d/d alleles, for 24 days and examined the effects on AHR activity, intestinal microbial community structure, inflammatory status, and response to chemically induced colitis. Cecal microbial community structure and metabolic potential were segregated according to host dietary and AHR status. Dietary broccoli associated with heightened intestinal AHR activity, decreased microbial abundance of the family Erysipelotrichaceae, and attenuation of colitis. In summary, broccoli consumption elicited an enhanced response in ligand-sensitive Ahr^b/b mice, demonstrating that in part the beneficial aspects of dietary broccoli upon intestinal health are associated with heightened AHR activity.

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 Ahr^b/b (high-affinity) or Ahr^d/d alleles (low-affinity) (Nebert & Bausserman, 1970). Mice harboring the Ahr^d/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.