Diet-induced expansion of pathobionts in experimental colitis

Abstract

Evidence presented in our recent study and elsewhere suggests that the interplay of dietary macronutrients with the intestinal lumen alters the microbial environment, and thus host-microbe interactions, in ways that are not always in a favorable, mutualistic fashion. Specifically, in inflammatory bowel diseases (IBD), experimental and clinical observations have implicated a detrimental impact of environmental/microbial factors on the etiopathogenesis of IBD in individuals with a background of genetic susceptibility to the disease.¹ Thus, now more than ever, we are realizing that specific intestinal microbes can metabolize and react to a wide array of dietary compositions that, in turn, markedly alter microbial populations. We aimed to understand if certain dietary fats that are prevalent in Western diets are capable of precipitating colonic inflammation through their actions on the enteric microbiota. On a background of genetic susceptibility, these microbial changes can impact host immune homeostasis and increase risk for disease. Here we elaborate on our findings and their potential implications for future medical care.

Keywords: :

• diet
• microbiota
• bile acids
• pathobiont
• colitis
• inflammation
• therapeutics

Inflammatory bowel diseases (IBD), as well as other immune-related human disorders, are relatively “new” diseases in that their incidence has increased significantly over the last half century, particularly in modern urban populations.^2-4 The rapidity of these developments are not likely caused by genetic drift, but by exposure to non-genetic factors introduced through changes in the environment and lifestyle of genetically susceptible individuals, triggering aberrant host responses that lead to IBD. In particular, diet is thought to play an important role in the increase of complex autoimmune and inflammatory disorders in Western countries. In our recent study, we examined if specific dietary fats prevalent in Western diets are capable of either precipitating or preventing/ameliorating colonic inflammation through their actions on the enteric microbiota.⁵ We hypothesized that on a background of genetic susceptibility, these microbial changes can impact host immune homeostasis and increase risk for disease.

Our results provide mechanistic insight into how an environmental factor such as the diet can profoundly influence the course of disease by altering the enteric microbial composition—particularly of low-abundance species. Bilophila wadsworthia, a low-abundant, sulfite-reducing anaerobe, “bloomed” on a high milk fat-based saturated fat diet, but not on other saturated fats such as lard, nor on polyunsaturated fat like safflower oil. The outcome was inflammation and/or overt colitis in every parameter tested. These findings suggest two things about future study design: First we can no longer simply study “high” vs. “low” fat diets- composition matters, because not all fats are made of the same fatty acids, not all carbohydrates the same polysaccharides and not all proteins the same amino acids. Second, in terms of human microbiome research, it is no longer sufficient to focus on phyla shifts in a given context—the low-abundance species are capable of more function than was previously thought.⁶ Furthermore, studies to-date investigating the impact of diet on the microbiome have focused primarily on direct effects of dietary substrates on the microbiota. These studies have revealed important information such as how bacteria metabolize indigestible fibers to produce short-chain fatty acids for example, which are critical to colonocyte health.^7,8 However, our findings precipitated a major dietary-influenced microbial alteration through no direct diet-microbe interaction. The primary impact of the saturated milk fat lies in the unique fatty acid composition consisting of high levels of hydrophobic stearate that places demand on the host for efficient emulsification by bile salts. The body adapts and shifts the composition of bile toward a greater taurocholate:glycocholate ratio—taurocholate being the much more efficient emulsifier for hydrophobic fats.^9-11 This is the key to the story. When taurocholate was increased (i.e., 80% taurocholate: 20% glycocholate) in the bile, B. wadsworthia bloomed, and when taurocholate was present at low-saturated fat levels (i.e., ~50% glycocholate: 50% taurocholate) B. wadsworthia was undetectable. Such considerations must be made when considering diet design for studies of the microbiota. The necessity of taurocholate to B. wadsworthia proliferation, rather than diet directly, was tested and confirmed in two important manners. First, we eliminated the effect of the diet by feeding mice only the low-fat diet, but gavaged daily with either glycocholate, taurocholate, or PBS. In specific pathogen free (SPF) mice, only the mice gavaged with taurocholate developed a bloom of B. wadsworthia. In germ-free mice, we took a closer look at B. wadsworthia in isolation by monoassociating it and again, only feeding mice a low-fat diet. When gavaged with glycocholate, taurocholate or PBS, only in mice gavaged with taurocholate was B. wadsworthia able to survive. Second, when bile was collected from the gall bladders of SPF mice consuming the low-fat, polyunsaturated or milk fat diet and added to taurine-free growth media, B. wadsworthia’s growth in vitro was significantly greater and more rapid in media containing bile from the milk fat-fed mice. It cannot be emphasized enough that in many cases, an observed microbial change may be a secondary effect of altering host physiology.

This aspect of the story emphasizes the profound impact a single dietary factor can have on our bodies, but also calls for deeper understanding of these dietary factors both natural and processed. For example, an important point has been misconstrued about this study. Since its publication, many have jumped to the conclusion that dairy, specifically milk, is the culprit in the rising incidence of IBD. It is necessary to point out that we were studying just the fat component of the milk and not the whole milk product containing all the proteins, carbohydrates, vitamins and minerals. Furthermore, our milk-fat was anhydrous milk-fat, which is a highly processed form of fat that is separated from milk and concentrated, almost like butter. Thus, the saturated fat content of this type of fat is greater than what you would find in even full-fat milk. We chose this source of fat because of its highly saturated nature (just as we chose safflower oil for its highly unsaturated nature) and because it represents the type of highly processed fat used in Western food manufacturing, particularly of confectionary foods such as ice creams and chocolates. Interestingly, we have received numerous anecdotal accounts from individuals who believe milk may be the source of theirs or their children’s recurring bouts of bowel inflammation, with a significant portion of the accounts related to antibiotic-treated or pasteurized milk. This raises some important questions to consider about the human diet as we move forward in studying host-diet-microbe interactions. We are just beginning to grasp how exquisitely sensitive the enteric microbiota are to dietary changes—not just at the broad macronutrient level, but at the specific composition level of those macronutrients. On the surface, these seem like too many variables to control, but such rigorously designed diet studies are feasible and necessary as we consider future therapies.

Downstream of these dietary effects on the host, the induced pathobiont expansion results in further impact on the host by inducing intestinal inflammation. This inflammation acts through canonical activation of dendritic cells presenting B. wadsworthia antigen to naive T cells. The unique aspect of this is that B. wadsworthia antigen specifically directs a T_H1 response. This response was defined by the elevated levels of IFNγ in the distal colonic mucosa and mesenteric lymph nodes only in mice consuming the milk-fat diet or gavaged with taurocholate, and through a series of ex vivo and in vitro immune assays. The specific TH1 response was further defined by the complete absence of IL-17 and IL-23 in monoassociated mice and very low levels in SPF mice. We determined that neither diet nor bile were having direct effects on this immune response because no inflammation was observed in germ-free mice consuming the milk-fat diet or gavaged with taurocholate in the absence of B. wadsworthia. The primary models used were indeed deficient in IL-10 production, thus opening the door for an aberrant immune response, however the observation that the MF diet still resulted in a bloom of B. wadsworthia in wild-type mice with an intact immune system suggests the necessity of a genetic pre-disposition to disease. Thus, our findings cannot be extrapolated to apply to all individuals consuming a diet rich in milk-fat. However, it does provide rationale for tailored interventions and a move toward personalized medicine. At the broad level, a patient with a family history of disease or with a known mutation should be treated with different interventions than a “wild-type” patient. For each individual, the microbiome, and in particular the “fingerprint” of the species that comprise it, may serve as effective therapeutic targets. As we begin to elucidate the factors that stimulate the commensal to pathogen switch and the pathways that carry out their virulence, we may discover ways to specifically suppress or eliminate a microbe by manipulating the unique enteric environment of the host. This may involve targeted pharmacotherapy that works synergistically with dietary manipulations.

Abbreviations:
IBD	=	inflammatory bowel diseases
B. wadsworthia	=	Bilophila wadsworthia
SPF	=	specific pathogen free, TH1, T cell helper 1
IFNγ	=	interferon gamma
IL-17	=	interleukin 17
IL-23	=	interleukin 23
IL-10	=	interleukin 10

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.