Colonic aberrant crypt formation accompanies an increase of opportunistic pathogenic bacteria in C57BL/6 mice fed a high-fat diet

Abstract

The increasing worldwide incidence of colon cancer has been linked to obesity and consumption of a high-fat Western diet. To test the hypothesis that a high-fat diet (HFD) promotes colonic aberrant crypt (AC) formation in a manner associated with gut bacterial dysbiosis, we examined the susceptibility to azoxymethane (AOM)-induced colonic AC and microbiome composition in C57/BL6 mice fed a modified AIN93G diet (AIN, 16% fat, energy) or an HFD (45% fat, energy) for 14 weeks. Mice receiving the HFD exhibited increased plasma leptin, body weight, body fat composition and inflammatory cell infiltration in the ileum compared with those in the AIN group. Consistent with the gut inflammatory phenotype, we observed an increase in colonic AC, plasma interleukin-6, tumor necrosis factor-α, monocyte chemoattractant protein-1 and inducible nitric oxide synthase in the ileum of the HFD-AOM group compared with the AIN-AOM group. Although the HFD and AIN groups did not differ in bacterial species number, the HFD and AIN diets resulted in different bacterial community structures in the colon. The abundance of certain short-chain fatty acid (SCFA) producing bacteria (e.g., Barnesiella) and fecal SCFA (e.g., acetic acid) content were lower in the HFD-AOM group compared with the AIN and AIN-AOM groups. Furthermore, we identified a high abundance of Anaeroplasma bacteria, an opportunistic pathogen in the HFD-AOM group. Collectively, we demonstrate that an HFD promotes AC formation concurrent with an increase of opportunistic pathogenic bacteria in the colon of C57BL/6 mice.

Introduction

Colon cancer is a major public health issue in the United States, with approximately 137,000 new cases and 50,000 deaths per year [1]. The increasing worldwide incidence of colon cancer has been linked to obesity and consumption of a high-fat (HF) Western diet [2], [3], [4]. Meta-analyses indicate that total energy intake is associated with a higher risk of colon cancer [5], [6]. The prevalence of obesity has increased drastically in the Western world as well as the regions where obesity was previously thought to be uncommon (e.g., China, South Korea) during recent decades [3], [4], [5], [6]. This global obesity epidemic has, in part, been attributed to the adoption of Western lifestyle, including increased consumption of a high-energy diet such as the high-fat diet (HFD) [5], [6], [7].

There is strong epidemiologic evidence linking diet-induced obesity with increased risk of colon cancer [5], [6], [7], [8]. Mechanistically, consumption of an HFD can lead to accumulation of excess body fat that is associated with adipose tissue dysfunction and a chronic state of low-grade inflammation known to promote tumor development [9], [10]. Studies in which germ-free mice inoculated with stool from tumor-bearing mice developed more tumors than those inoculated with stool from tumor-free mice support a causal role for the gut microbiota in colon carcinogenesis in an azoxymethane (AOM)-induced colon cancer model [11]. A link between the gut microbiome and colon cancer is increasingly apparent, and an altered microbiota is found among individuals with colon cancer [12], [13]. For example, the Clostridium, Roseburia and Eubacterium spp. are less prevalent in colon cancer subjects than in healthy individuals [14]. In addition, some strains of Bacteroides fragilis and Escherichia coli are enterotoxigenic and directly promote tumorigenesis [11], [15].

The symbiotic relationship of hundreds of microbial species with the host requires a tuned response to prevent host damage, e.g., inflammation, while supporting the presence of the potentially beneficial microbes. The colonic microbiota is critical for mucosal tolerance under normal conditions and prevents pathogen infections [16], [17]. Recent studies show that immunological processes participate in the maintenance of homeostasis with the microbiota and that disturbance of host immunity or the microbial ecosystem results in disease-provoking dysbiosis [16], [17]. The gastrointestinal tract is poised in a state of equilibrium that permits rapid protective responses against pathogens, but curtails damage by hindering long-lasting vigorous inflammatory processes [18]. While the pathways active in promoting obesity-related colon cancer remain to be characterized, it is possible that the process may involve the colonic microbiota, which affects gut inflammatory status and the extraction of energy from the diet [19], [20], [21]. It is known that dietary fiber type, fat composition, composition of gut microbiome/metabolites, animal species and experimental time point are the major factors contributing to the development of inflammation and colon cancer. However, there are few comprehensive studies including all above factors in the context of HFD-induced obesity. It remains largely unknown how colonic aberrant crypt (AC, putative preneoplastic lesions) formation affects gut microbiome/metabolite composition.

We hypothesize that not only does an HFD promote AC formation but obesity and AC formation are associated with bacterial dysbiosis in the colon. With a comprehensive manner, we tested this hypothesis using the HFD-induced obesity in an AOM mouse model.