Gnotobiotic animals are raised in germ-free environments—with no exposure to microbes—and then colonized at specific stages of life with specified microbial communities. Gnotobiotic mice provide an excellent system for controlling host genotype, microbial community composition, diet, and housing conditions. Microbial communities harvested from donor mice with defined genotypes and phenotypes can be used to determine how the donors’ microbial communities affect the properties of formerly germ-free recipients. The recipients may also affect the transplanted microbiota and its microbiome. Thus gnotobiotic mice afford investigators an opportunity to marry comparative studies of donor communities to functional assays of community properties and to determine how (and for how long) these functions influence host biology.
The cardiovascular system
The gut microbiota affects the elaborate microvasculature underlying the small-intestinal epithelium: capillary network density is markedly reduced in adult germ-free animals but can be restored to normal levels within 2 weeks after gut microbiota transplantation. Mechanistic studies have shown that the microbiota promotes vascular remodeling in the gut through effects on a novel extravascular tissue factor–protease-activated receptor (PAR1) signaling pathway. Heart weight measured echocardiographically or as wet mass and normalized to tibial length or lean body weight is significantly reduced in germ-free mice; this difference is eliminated within 2 weeks after colonization with a gut microbiota. During fasting, a gut microbiota–dependent increase in hepatic ketogenesis (regulated by peroxisome proliferator–activated receptor α) occurs, and myocardial metabolism is directed to ketone body utilization. Analyses of isolated, perfused working hearts from germ-free and colonized animals, together with in vivo assessments, have shown that myocardial performance in germ-free mice is maintained by increasing glucose utilization. However, heart weight is significantly reduced in both fasted and fed mice; this heart-mass phenotype is completely reversed in germ-free mice fed a ketogenic diet. These findings illustrate how the gut microbiota benefits the host during periods of nutrient deprivation and represent one link between gut microbes and cardiovascular metabolism and health.
Conventionally raised apoE-deficient mice develop a less severe form of atherosclerosis than their germ-free counterparts when fed a high-fiber diet. This protective effect of the microbiota is obviated when animals are fed a diet low in fiber and high in simple sugars and fat. A number of the beneficial effects attributed to diets with high proportional representation of whole grains, fruits, and vegetables are thought to be mediated by end products of microbial metabolism of dietary compounds, including short-chain fatty acids and metabolites derived from flavonoids. Conversely, microbes can convert otherwise harmless dietary compounds into metabolites that increase risk for cardiovascular disease. Studies of mice and human volunteers have revealed that gut microbiota metabolism of dietary L-carnitine, which is present in large amounts in red meat, yields trimethylamine-N-oxide, which can accelerate atherosclerosis in mice by suppressing reverse cholesterol transport.
Yet another facet of microbial influence on cardiovascular physiology was revealed in a study of mice deficient in Olfr78 (a G protein–coupled receptor expressed in the juxtaglomerular apparatus, where it regulates renin secretion in response to short-chain fatty acids) or Gpr41 (another short-chain fatty acid receptor that, together with Olfr78, is expressed in the smooth muscle cells present in small resistance vessels). This study demonstrated that the microbiota can modulate host blood pressure via short-chain fatty acids produced by microbial fermentation.
Adult germ-free mice have greater bone mass than their conventionally raised counterparts. This increase in bone mass is associated with reduced numbers of osteoclasts per unit bone surface area, reduced numbers of CD11b+/GR1 osteoclast precursors in bone marrow, decreased numbers of CD4+ T cells, and reduced levels of expression of the osteolytic cytokine tumor necrosis factor α. Colonization with a normal gut microbiota resolves these observed differences between germ-free and conventionally raised animals.
Adult germ-free and conventionally raised mice differ significantly in levels of 38 out of 196 identified cerebral metabolites, 10 of which have known roles in brain function; included in the latter group are N-acetylaspartic acid (a marker of neuronal health and attenuation), pipecolic acid (a presynaptic modulator of γ-aminobutyric acid levels), and serine (an obligatory co-agonist at the glycine site of the N-methyl-d-aspartate receptor). Propionate, a short-chain fatty acid product of gut microbial-community metabolism of dietary fiber, affects expression of genes involved in intestinal gluconeogenesis via a gut–brain neural circuit involving free fatty-acid receptor 3; this effect provides a mechanistic explanation for the documented beneficial impact of dietary fiber in enhancing insulin sensitivity and reducing body mass and adiposity.
Studies of a mouse model (maternal immune activation) with stereotyped/repetitive and anxiety-like behaviors indicate that treatment with a member of the human gut microbiota, Bacteroides fragilis, corrects gut barrier (permeability) defects; reduces elevated levels of 4-ethylphenylsulfate, a metabolite seen in the maternal immune activation model that has been causally associated with the animals’ behavioral phenotypes; and ameliorates some behavioral effects. These observations highlight the importance of further exploration of potentially co-evolved relationships between the microbiota and host behavior.
Many foundational studies have shown that the gut microbiota plays a key role in the maturation of the innate as well as the adaptive components of the immune system. The intestinal epithelium, which is composed of four principal cell lineages (enterocytes plus goblet, Paneth, and enteroendocrine cells), acts as a physical and functional barrier to microbial penetration. Goblet cells produce mucus that overlies the epithelium, where it forms two layers: an outer (luminal-facing) looser layer that harbors microbes and a denser lower layer that normally excludes microbes. Members of the Paneth cell lineage reside at the base of crypts of Lieberkühn and secrete antimicrobial peptides. Studies in mice have demonstrated that Paneth cells directly sense the presence of a microbiota through expression of the signaling adaptor protein MyD88, which helps transduce signals to host cells upon recognition of microbial products through Toll-like receptors (TLRs). This recognition drives expression of antibacterial products (e.g., the lectin RegIIIγ) that act to prevent microbial translocation across the gut mucosal barrier.
The intestine is enriched for B cells that produce IgA, which is secreted into the lumen; there it functions to exclude microbes from crossing the mucosal barrier and to restrict dissemination of food antigens. The microbiota plays a key role in development of an IgA response: germ-free mice display a marked reduction in IgA+ B cells. The absence of a normal IgA response can lead to a massive increase in bacterial load. B cell–derived IgA that targets specific members of the gut microbiota plays an important role in preventing activation of microbiota-specific T cells.
Gut bacterial species elicit development of protective TH17 and TH1 responses that help ward off pathogen attack. Members of the microbiota also promote the development of a specialized population of CD4+ T cells that prevent unwarranted inflammatory responses. These regulatory T cells (Tregs) are characterized by expression of the transcription factor forkhead box P3 (FOXP3) and by expression of other cell-surface markers. There is a paucity of Tregs in the colonic lamina propria of germ-free mice. Specific members of the microbiota—including a consortium of Clostridium strains isolated from the mouse and human gut as well as several human-gut Bacteroides species —expand the Treg compartment and enhance immunosuppressive functions.
The microbiota is a key trigger in the development of inflammatory bowel disease (IBD) in mice that harbor mutations in genes associated with IBD risk in humans. Moreover, components of the gut microbiota can modify the activity of the immune system to ameliorate or prevent IBD. Mice containing a mutant ATG16L1 allele linked to Crohn’s disease are particularly susceptible to IBD. Upon infection with mouse norovirus and treatment with dextran sodium sulfate, expression of a hypomorphic ATG16L1 allele leads to defects in small-intestinal Paneth cells and renders mice significantly more susceptible to ileitis than are wild-type control animals. This process is dependent on the gut microbiota and highlights how the intersection of host genetics, infectious agents, and the microbiota can lead to severe immune pathology; i.e., the pathogenic potential of a microbiota may be context-dependent, requiring a confluence of factors. An important observation is that members of the gut microbiota, including B. fragilis or members of Clostridium, prevent the severe inflammation that develops in mouse models mimicking various aspects of human IBD.
The gut microbiota has been implicated in promoting immunopathology outside of the intestine. Multiple sclerosis develops in conventionally raised mice whose CD4+ T cell compartment is reactive to myelin oligodendrocyte protein; their germ-free counterparts are completely protected from development of multiple sclerosis–like symptoms. This protection is reversed by colonization with a gut microbiota from conventionally raised animals.
Inflammasomes are cytoplasmic multiprotein complexes that sense stress and damage-associated patterns. Mice deficient in NLRP6, a component of the inflammasome, are more susceptible to colitis induced by administration of dextran sodium sulfate. This enhanced susceptibility is associated with alterations in the gut microbiota of these animals relative to that of wild-type controls. Mice are coprophagic, and co-housing of NLRP6-deficient mice with wild-type mice is sufficient to transfer the enhanced susceptibility to colitis induced by dextran sodium sulfate. Similar findings have been reported for mice deficient in the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain). ASC-deficient mice are more susceptible to the development of a model of nonalcoholic steatohepatitis. This susceptibility is associated with alterations in gut microbiota structure and can be transferred to wild-type animals by co-housing.
Germ-free mice are resistant to diet-induced obesity. Genetically obese ob/ob mice have gut microbial-community structures that are profoundly altered from those in their lean wild-type (+/+) and heterozygous +/ob littermates. Transplantation of the ob/ob mouse microbiota into wild-type germ-free animals transmits an increased-adiposity phenotype not seen in mice receiving microbiota transplants from +/+ and +/ob littermates. These differences are not attributable to differences in food consumption but rather are associated with differences in microbial community metabolism. Roux-en-Y gastric bypass produces pronounced decreases in weight and adiposity as well as improved glucose metabolism—changes that are not ascribable simply to decreased caloric intake or reduced nutrient absorption. 16S rRNA analyses have documented that changes in the gut microbiota after this surgery are conserved among mice, rats, and humans; animal studies have demonstrated these changes along the length of the gut but most prominently downstream of the site of surgical manipulation of the bowel. Notably, transplantation of the gut microbiota from mice that have undergone Roux-en-Y gastric bypass to germ-free mice that have not had this surgery produces reductions in weight and adiposity not seen in recipients of microbiotas from mice that underwent sham surgery.
The gut microbiota confers protection against the development of type 1 diabetes mellitus in the non-obese diabetic (NOD) mouse model. Disease incidence is significantly lower in conventionally raised male NOD mice than in their female counterparts, while germ-free males are as susceptible as their female counterparts. Castration of males increases disease incidence, while androgen treatment of females provides protection. Transfer of the gut microbiota from adult male NOD mice to female NOD weanlings is sufficient to reduce the severity of disease relative to that among females receiving a microbiota from an adult female or an unmanipulated female. The blocking of protection by treatment with flutamide highlights a functional role for testosterone signaling in this microbiota-mediated protection against type 1 diabetes.
NOD mice deficient in MyD88, a key component of the TLR signaling pathway, do not develop diabetes and exhibit increased relative abundance of members of the family-level taxon Lactobacillaceae. Consistent with these findings, investigators have documented lower levels of representation of members of the genus Lactobacillus in children with type 1 diabetes than in healthy controls. Components of lactobacilli have been shown to promote gut barrier integrity. Studies in various animal models indicate that translocation of bacterial components, including bacterial lipopolysaccharides, across a leaky gut barrier triggers low-grade inflammation, which contributes to insulin resistance. Mice deficient in TLR5 exhibit alterations in the gut microbiota and hyperphagia, and they develop features of metabolic syndrome, including hypertension, hyperlipidemia, insulin resistance, and increased adiposity.
The gut microbiota regulates biosynthesis as well as metabolism of host-derived products; these products can signal through host receptors to shape host physiology. An example of this symbiosis is provided by bile acids, which direct metabolic effects that are largely mediated through the farnesoid X receptor (FXR, also known as NR1H4). In leptin-deficient mice, FXR deficiency protects against obesity and improves insulin sensitivity. In mice with diet-induced obesity that are subjected to vertical sleeve gastrectomy, the surgical procedure results in elevated levels of circulating bile acids, changes in the gut microbiota, weight loss, and improved glucose homeostasis. However, weight reduction and improved insulin sensitivity are mitigated in animals with engineered FXR-deficiency.
Evidence is accumulating that pharmacogenomic studies need to consider the gene repertoire present in our H. sapiens genome as well as that in our microbiomes. For example, digoxin is inactivated by the human gut bacterium Eggerthella lenta, but only by strains with a cytochrome-containing operon. Expression of this operon is induced by digoxin and inhibited by arginine. Studies in gnotobiotic mice established that dietary protein affects (reduces) microbial metabolism of digoxin, with corresponding alterations in levels of the drug in both serum and urine. These findings reinforce the need to consider strain-level diversity in the gut microbiota when examining interpersonal variations in the metabolism of orally administered drugs.
Characterizing the effects of the human microbiota on host biology in mice and humans
Questions about the relationship between human microbial communities and health status can be posed in the following general format: Is there a consistent configuration of the microbiota definable in the study population that is associated with a given disease state? How is the configuration affected by remission/relapse or by treatment? If a reconfiguration does occur with treatment, is it durable? How is host biology related to the configuration or reconfiguration? What is the effect size? Are correlations robust to individuals from different families and communities representing different ages, geographic locales, and lifestyles?
As in all studies involving human microbial ecology, the issue of what constitutes a suitable reference control is extremely important. Should we choose the person himself or herself, family members, or age- or gender-matched individuals living in the same locale and representing similar cultural traditions? Critically, are the relationships observed between microbial community structure and expressed functions a response to disease state (i.e., side effects of other processes), or are they a contributing cause? In this sense, we are challenged to evolve a set of Koch’s postulates that can be applied to whole microbial communities or components of communities rather than just to a single purified organism. As in other circumstances in which experiments to determine causality of human disease are difficult or unethical, Hill’s criteria, which examine the strength, consistency, and biologic plausibility of epidemiologic data, can be useful.
Sets of mono- and dizygotic twins and their family members represent a valuable resource for initially teasing out relationships between environmental exposures, genotypes, and our own microbial ecology. Similarly, monozygotic twins discordant for various disease states enhance the ability to determine whether various diseases can be linked to a person’s microbiota and microbiome. A twin-pair sampling design rather than a conventional unrelated case–control design has advantages owing to the pronounced between-family variability in microbiota/microbiome composition and the potential for multiple states of a community associated with disease. Transplantation of a microbiota from suitable human donor controls representing different disease states and communities (e.g., twins discordant for a disease) to germ-free mice is helpful in establishing a causal role for the community in pathogenesis and for providing insights relevant to underlying mechanisms. In addition, transplantation provides a preclinical platform for identifying next-generation probiotics, prebiotics, or combinations of the two (synbiotics). Obesity and obesity-associated metabolic dysfunction illustrate these points.
The gut microbiotas (and microbiomes) of obese individuals are significantly less diverse than those of lean individuals; the implication is that there may be unfilled niches (unexpressed functions) that contribute to obesity and its associated metabolic abnormalities. Le Chatelier and colleagues observed a bimodal distribution of gene abundance in their analysis of 292 fecal microbiomes: low-gene-count (LGC) individuals averaged 380,000 microbial genes per gut microbiome, while high-gene-count (HGC) individuals averaged 640,000 genes. LGC individuals had an increased risk for type 2 diabetes and other metabolic abnormalities, whereas the HGC group was metabolically healthy. When gene content was used to identify taxa that discriminated HGC and LGC individuals, the results revealed associations between anti-inflammatory bacterial species such as Faecalibacterium prausnitzii and the HGC group and between proinflammatory species such as Ruminococcus gnavus and the LGC group. LGC microbiomes had significantly greater representation of genes assigned to tricarboxylic acid cycle modules, peroxidases, and catalases—an observation suggesting a greater capacity to handle oxygen exposure and oxidative stress; HGC microbiomes were enriched in genes involved in the production of organic acids, including lactate, propionate, and butyrate—a result suggesting increased fermentative capacity.
Transplantation of an uncultured fecal microbiota from twins stably discordant for obesity or of bacterial culture collections generated from their microbiota transmits their discordant adiposity phenotypes as well as obesity-associated metabolic abnormalities to recipient germ-free mice. Co-housing of the recipient coprophagic gnotobiotic mice results in invasion of specific bacterial species from the transplanted lean twin’s culture collection into the guts of cage mates harboring the obese twin’s culture collection (but not vice versa), thereby preventing the latter animals from developing obesity and its associated metabolic abnormalities. It is noteworthy that invasion and prevention of obesity and metabolic phenotypes are dependent on the type of human diets fed to animals: prevention is associated with a diet low in saturated fats and high in fruit and vegetable content, but not with a diet high in saturated fats and low in fruit and vegetable content.
This approach provides evidence for a causal role for the microbiota in obesity and its attendant metabolic abnormalities. It also provides a method for defining unoccupied niches in disease-associated microbial communities, the role of dietary components in determining how these niches can be filled by human gut–derived bacterial taxa, and the effects of such occupancy on microbial and host metabolism. It also provides a way to identify health-promoting diets and next-generation probiotics representing naturally occurring members of our indigenous microbial communities that are well adapted to persist in a given body habitat.
A key to this approach is the ability to harvest a microbial community from a donor representing a physiology, disease state, lifestyle, or geography of interest; to preserve the donor’s community by freezing it; and then to resurrect and replicate it in multiple recipient gnotobiotic animals that can be reared under conditions where environmental and host variables can be controlled and manipulated to a degree not achievable in clinical studies. Since these mice can be followed as a function of time prior to and after transplantation, in essence, a snapshot of a donor’s community can be converted into a movie. Transplantation of intact uncultured human (fecal) microbiota samples from multiple donors representing the phenotype of interest, with administration of the donors’ diets (or derivatives of those diets) to different groups of mice, is one way to assess whether transmissible responses are shared features of the microbiota or are highly donor specific. A second step is to determine whether the culturable component of a representative microbiota sample can transmit the phenotype(s) observed with the intact uncultured sample. Possession of a collection of cultured organisms that have co-evolved in a given donor’s body habitat sets the stage for the selection of subsets of the collection for testing in gnotobiotic mice, the determination of which members are responsible for effecting the phenotype, and the elucidation of the mechanisms underlying these effects. The models used may inform the design and interpretation of clinical studies of the very individuals and populations whose microbiota are selected for creating these models.
Human-to-human fecal microbiota transplantation (FMT) is currently the most direct way to establish proof-of-concept for a causal role for the microbiota in disease pathogenesis. A human donor’s feces are provided to a recipient via nasogastric tube or another technique. Numerous small trials have documented the effects of FMT from healthy donors to recipients with diseases ranging from C. difficile infection to Crohn’s disease, ulcerative colitis, and type 2 diabetes. Only a few of these studies have used a double-blind, placebo-controlled design.
In a double-blind, controlled trial involving men 21–65 years old with a body mass index of >30 kg/m2 and documented insulin resistance, FMT was performed using a microbiota from metabolically healthy lean donors or from the study participants themselves. A microbiota from lean donors significantly improved peripheral insulin sensitivity over that in controls. This change was associated with an increase in the relative abundance of the butyrate-producing bacteria related to Roseburia intestinalis (in the feces) and Eubacterium hallii (in the small intestine).
The efficacy of FMT for the treatment of recurrent C. difficile infection has been assessed in a number of small trials. One unblinded, placebo-controlled trial assessed the use of FMT in 42 patients with recurrent C. difficile infection (defined as at least one relapse after treatment with vancomycin or metronidazole for ≥10 d). Patients were pretreated with oral vancomycin. The experimental group then received FMT via nasoduodenal tube from healthy volunteer donors (<60 years of age) selected from the community. Controls underwent sterile lavage or received oral vancomycin alone. In 10 weeks of follow-up, infection was cured (with cure defined as three negative fecal tests for C. difficile toxin) in 81% of patients in the FMT group (13 of 16) but in only 23% (3 of 13) in the bowel-lavage control arm and 31% (4 of 13) in the vancomycin-only group. Metagenomic analysis of microbiota samples collected before and after treatment revealed an increased representation of Bacteroidetes and Clostridium clusters IV and XIVa, along with a 100-fold decrease in the relative abundance of Proteobacteria, in the FMT group.
A meta-analysis of FMT in C. difficile infection examined 20 case-series publications, 15 case reports, and the one unblinded study described above. All but one of these studies used fresh (not frozen) fecal samples. Donor selection varied, although most donors were family members or relatives and most studies excluded donors who had recently received antibiotics. It is noteworthy that the concentrations of infused donor feces varied widely (i.e., from 5 g to 200 g, resuspended in 10–500 mL); these fecal suspensions were introduced at different sites along the gastrointestinal tract, including the stomach and points throughout the small intestine and colon. Resolution of infection, which was frequently assessed on the basis of symptom resolution (with C. difficile toxin testing rarely performed), was documented in 87% (467) of 536 treated patients. The most common adverse events reported were diarrhea (94% of cases) and abdominal cramps (31%) on the day of infusion. The meta-analysis was limited to clinical outcomes and did not specifically address the role of the microbiota in disease resolution (e.g., the extent of invasion of donor taxa; their persistence; or the long-term effects of transplantation on various facets of host biology, which generally have not been evaluated).
Sober and thoughtful consideration needs to be applied to the therapeutic use of FMT, which represents an early and rudimentary approach to microbiota manipulation that very likely will be replaced by administration of defined collections of sequenced, cultured members of the human microbiota (probiotic consortia). A number of published reports on FMT have garnered significant public attention. This attention, coupled with an increasing public appreciation of the beneficial nature of our interactions with microbes, demands that the precautionary principle be honored and that risks versus benefits of such interventions be carefully evaluated.
To date, most FMT trials have failed to define (or have differed in) significant confounders, including (1) the criteria used for donor sample selection; (2) the methods used for donor sample preparation and characterization as well as the decision about whether or not to create a repository for donor and recipient samples that will permit retrospective analyses (and meta-analyses for given disease states); (3) the development of minimal standards for assessing the invasion of recipient gut communities by taxa from donor microbiota (using microbial source-tracking methods) as well as the timing, duration, nature, and breadth of sampling of the recipient as a function of transplantation; (4) the adoption of minimal standards for collection of patients’ clinical data (e.g., age, diet, antibiotic use) and the establishment of databases for entering these data (including use of a defined vocabulary for annotating the clinical data); and (5) the development of standards for informed consent in lieu of knowledge of the long-term effects of the procedure. The regulatory landscape is evolving. The U. S. Food and Drug Administration recently issued an enforcement policy specifically addressing the use of FMT for the treatment of recurrent C. difficile infection; this policy indicates that the agency intends to “exercise enforcement discretion regarding the investigational new drug (IND) requirements for the use of FMT to treat C. difficile infection not responding to standard therapies,” but it does not waive IND requirements for other FMT studies.