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Tuesday, January 12, 2016
Advances in Human Microbiome Science: Intestinal Diseases
Organizers: Mercedes Beyna (Pfizer), John Hambor (Boehringer Ingelheim), Nilufer Seth (Pfizer), Erick Young (Boehringer Ingelheim), and Sonya Dougal (The New York Academy of Sciences) Keynote Speaker: Stanislav Dusko Ehrlich (French National Institute for Agricultural Research; King's College London)Presented by the Microbiome Science Discussion Group
Reported by Hannah Rice | Posted December 22, 2015
On October 15, 2015, the Academy's Microbiome Science Discussion Group convened researchers for Advances in Human Microbiome Science, the first of three symposia on the causal relationships between microbiota and disease—this one focused on intestinal diseases. Human colon microbiota form one of the densest bacterial ecosystems, with 100 trillion microorganisms, or tenfold more cells than the body itself. These commensal microbiota have beneficial health effects such as aiding digestion, resisting pathogen invasion, and producing useful metabolites, but dysbiotic bacterial ecosystems or dysregulated host immunity can stimulate inflammatory responses that lead to disease.
The Crohn's & Colitis Foundation of America reported in 2014 that 1.6 million people in the U.S. had inflammatory bowel disease (IBD), a condition that increases the risk of intestinal bleeding, weight loss, life-threatening infection, and colorectal cancer. There is evidence that the gut microbiota are implicated in other chronic diseases, including arthritis, diabetes, obesity, allergies, cancers, and HIV. Researchers hope that microbiome research will yield insights into disease susceptibility and treatment strategies. The meeting featured a keynote and two plenary sessions, as well as data-blitz and late-breaking data presentations by young investigators.
Stanislav Dusko Ehrlich of the French National Institute for Agricultural Research (INRA) and King's College London gave a keynote address describing how better understanding and management of the microbiome could reduce the burden of chronic diseases, yielding diagnostics and disease-monitoring tools and opportunities for disease prevention. Metagenomics projects such as MetaHIT have revealed tremendous genetic diversity among human microbiomes as well as many common genes that could provide a focus for clinical study. Scientists are working to understand how the microbiome influences health and disease and to uncover the roles played by specific microbes.
Metagenomics maps the genes in the microbiome, the other human genome. (Image courtesy of Stanislav Dusko Ehrlich)
Ehrlich and colleagues showed that liver cirrhosis can be diagnosed on the basis of finding 7 bacterial species, identified by gene clusters, in a stool sample. Metagenomic analysis also predicted disease progression: individuals at later stages had worse symptoms as well as a greater number of invasive species, which displace normal gut flora. Microbiome composition is probably the result of a combination of genes and environment. One in four people has a low-diversity microbiome, associated with weight gain, diabesity, and poorer health. Yet microbiota are responsive to intervention: microbial diversity increases when people stop smoking or eat a healthier diet. Ehrlich's team is looking for ways to target the microbiome to prevent disease. The researchers have found early success with a drug called DAV132, designed to protect the microbiota from the depleting effects of antibiotics.
An often-posed question is whether the microbiome is a cause or a consequence of chronic disease; Ehrlich suggested it may be neither, but instead a contributor that responds to or exacerbates other disease triggers. "We should strive to improve health by modulating an unhealthy or toxic microbiome," he said. "We need more prevention because today's medicine is curative."
There are probably a variety of dysbiotic gut ecosystems that can precede diseased or inflammatory states. (Image courtesy of Jonathan Braun)
In the first plenary session, Jonathan Braun of the University of California, Los Angeles, described another way to measure the microbiome—metabolite profiling. In IBD, which includes Crohn's disease and colitis, some affected individuals have a disease-associated metabolic signature while others have close-to-healthy metabolites. In otherwise healthy people, altered metabolites might indicate preclinical disease or could reflect a pre-disease ecosystem that confers heightened risk. To illustrate the importance of gene expression and function in microbiome analysis, Braun pointed to a study in mice showing that yoghurt ingestion—which introduced small quantities of new microbes and genes—had far-reaching metabolic effects throughout the gut microbiota.
Studies suggest gut ecosystems are not confined to be either healthy or dysbiotic; a variety compositions can progress to disease, and conversely, successful intervention can produce a "post-disease dysbiotic ecosystem" unlike the normal state. In a study of children with Crohn's disease and their families, Braun's team found disease-associated, healthy, and at-risk metabotypes (metabolite phenotypes). The researchers plan to monitor the last group for metabolic changes and progression to disease. The interaction of gut microbiota, genes, and metabolites may be important in disease susceptibility. Specifically, one proposal is that "the habitat is changed genetically, organisms change as a result, so [the] ecosystem changes and their [metabolic] products put you closer to the threshold of trouble," Braun explained. Metabolic profiling could indicate interventions for IBD and other conditions, such as replacing missing metabolites or blocking disease-associated products with drugs.
Mucosal inflammation is a genetic and microbial process that includes changes in the epithelial barrier, in bacterial control, in immune regulation, and in cellular responses. (Image presented by Jonathan Braun courtesy of Khor et al. Nature. 2011.)
Natural product drug discovery has traditionally used samples from soil and marine ecosystems, but the gut microbiota are a rich source of bioactive compounds. Michael Fischbach of the University of California, San Francisco, discussed his group's work to understand metabolic pathways in bacterial ecosystems and to find novel products. One method of drug discovery is to culture bacteria and screen metabolites for therapeutic activity. But this approach misses the compounds microbes synthesize to communicate with one another in their natural environments. Taking the reverse approach, Fischbach's lab computationally screens whole genomes to find biosynthetic gene clusters—groups of genes likely, on the basis of their similarity to known protein-coding genes, to code for proteins. The goal is to "intuit what [microbes] are capable of producing even [without knowing] under what conditions those molecules are made," he said. The researchers have designed methods to predict the chemical structures of metabolites from just the gene sequences.
Using data from the human microbiome project, the lab identified 600 biosynthetic gene clusters in the human gut microbiome, all encoding unknown proteins. Analysis of a family of gene clusters found in 90% of gut samples, some from bacteria that have never been isolated, yielded 33 new molecules. Fischbach emphasized the need to characterize these products to understand the effects of gut microbes on human health. Potent metabolites such as the thiopeptide antibiotics the researchers found genes for are often present in the gut at concentrations similar to those used for pharmaceutical drugs, and have substantial effects on host immunology and metabolism. "This is not subtle. This is a very concrete contribution ... to host biology," he said.
Although the immune system continually responds to invading pathogens, the commensal microbiota are relatively stable. Andy Goodman of Yale University School of Medicine studies this microbial resilience to the body's antimicrobial defenses. One such defense is the secretion of antimicrobial peptides (AMPs), which disrupt the bacterial cell membrane to cause cell death. Goodman's group found Bacteroides microbes, common in the human gut, to be highly resistant to AMPs, and identified a protective gene in the microbes called lpxF. The mechanism of the microbes' resistance to AMPs highlights the interplay among pathogens, gut microbiota, and the immune response. Experiments in mice showed that without the protective lpxF gene, Bacteroides in the gut are lost after mice are exposed to a pathogen that stimulates gut inflammation. But expose the mice to the pathogen altered so as not to produce inflammation, or expose lpxF-mutant cells to a pathogen directly in vitro, and there is no effect. The mutant microbes are thus sensitive to the host response to the perturbation, and wildtype microbes are immune to that response. In a complex microbial ecosystem such as the gut, the resilience of commensal species means that microbiome composition can return to normal after disruption. "Although we know a lot about how the host tolerates the microbiota, there are going to be more mechanisms like this of the microbiota tolerating the host that will be just as important for understanding the outcome of pathogen-induced inflammation and other types of inflammation," Goodman said.
The first session ended with a discussion of the role of microbiota as we age and whether it is possible to extend healthy life by manipulating the microbiome. Paul W. O'Toole of the University College Cork studies dietary and fecal-microbiome diversity in seniors living in the community and in long-term residential care. His team found that dietary diversity and microbiota diversity are correlated. Residents in long-term care had poorer dietary diversity, lower gene-count metagenomes, and depleted general metabolism, as well as higher incidence of geriatric depression, frailty, inflammation, and sarcopenia. The team linked the differences in depression, frailty, and inflammation with microbiota composition. The gut microbiota of seniors who moved from the community to long-term care resembled that of others in long-term care after 6 months to a year; to determine whether these changes in microbiota could be the result of illness, not diet, the team also assessed healthy community-living seniors with poor diets, who were found to have microbiomes similar to those of long-term care seniors, as well as similar frailty and cognitive test scores and similar IL-6 markers of immunity. These studies support the association between diet, microbiota, and health.
In the second plenary session, Rodolphe Clerval explained how Enterome uses metagenomics to find bacterial species and metabolic products associated with disease. The company has designed methods to quickly profile the microbiome of fecal and mucosal samples, using metagenomics tools developed by Ehrlich and others to build DNA libraries, and then cloning DNA segments into Escherichia coli and testing the bacteria for disease-associated biological activity, such as stimulation of regulatory T (Treg) cells. The researchers then sequence the genomes of clones of interest to identify the specific genes and compounds involved in the activity. The method is particularly useful for studying species that constitute a small proportion of the microbiota or cannot be cultured. Clerval noted the need to link genomic work like this, aimed at finding new drugs and targets, with diagnostic and biomarker studies in patients; for example, profiling the microbiomes of Crohn's disease patients at various stages of disease not only for biomarkers of disease but also for compounds that could be targeted or used therapeutically.
One of the protective effects of commensal microbiota is to activate and educate immune responses.R. Balfour Sartor and his team at the University of North Carolina School of Medicine showed that microbes induce immune cells in the colon to secrete interleukin 10 (IL-10)—a cytokine previously shown to prevent colitis—by demonstrating that IL-10 levels increase after bacteria colonize the gut in germ-free mice. Microbiota-induced secretion of IL-10 helps maintain homeostasis in the gut by mediating B-cell regulation of proinflammatory cytokines such as interferon-γ. After an immune response to an invading pathogen creates a spike in gut inflammation, the proinflammatory mechanisms are suppressed by IL-10, stimulated by commensal microbiota. Particular bacterial species involved in IL-10 secretion and regulatory activity of T cells can reverse inflammation, and thus could be part of a treatment strategy to increase protective immune responses in IBD.
Sarkis Mazmanian of the California Institute of Technology also discussed bacterial modulation of immune mechanisms via IL-10. Bacteroides fragilis are commonly found among human gut commensal bacteria and usually prevalent when present. Mazmanian and colleagues showed that these microbes secrete and deliver polysaccharide A (PSA) to dendritic cells via membrane vesicles. PSA, an antianflammatory molecule, then activates dendritic cells to signal naïve T cells to become Treg cells that produce IL-10. Other researchers have found that these PSA-mediated mechanisms may be involved in colon cancer and multiple sclerosis.
There may be no pathogen involved in IBD; instead, disease could be the result of a loss of immune tolerance to commensal microbes. Dysbiosis, an overgrowth of certain bacteria, a change in gene transcription, or a change bacterial location may trigger disease. (Image presented by Sarkis Mazmanian courtesy of Peterson et al. Cell Host Microbe. 2008.)
Mazmanian's group set out to discover how gene mutations known to increase colitis risk might be involved in PSA signaling. Some mutations associated with colitis are involved in autophagy pathways, and the researchers found that PSA activates these pathways in dendritic cells. When the genes are knocked out or mutated in animal models, PSA does not protect mice from colitis; and human dendritic cells with the same mutations do not respond to PSA signaling.
The stimulation of autophagy pathways could protect against colitis by clearing buildups of dysbiotic bacteria in the gut and preventing endoplasmic reticulum stress (linked to IBD), in addition to reducing inflammation via Treg activation. "The [autophagy] machinery has been coopted by bacteria ... to allow its signals to be transmitted to the host," Mazmanian said. Thus IBD patients with defects in autophagy may be unable to receive signals from symbiotic gut microbiota.
Some IBD treatments aim to heal inflamed mucosa in the intestine, repairing structural damage to the epithelial wall. Randy Longman of Weill Cornell Medical College described his lab's investigation of the microbial mechanisms involved in mucosal healing in IBD. Longman's team found that in Crohn's disease and colitis, innate lymphoid cells called ILC3s in the mucosa secrete a cytokine, IL-22, that stimulates epithelial cells to produce AMPs, promoting mucosal healing. Studies suggest that microbiota promote IL-22 production, and Longman's group found evidence that a signal is relayed from microbes to ILC3s via a type of mononuclear phagocyte (MNP) that produces ILC3-stimulating cytokines. The MNPs, called CX3CR1+ cells, can extend processes through the epithelial wall to sample gut microbiota; when there is dysbiosis, these cells carry gut bacteria to lymph nodes, triggering immunity to commensal microbes.
Longman closed with a discussion of the effects of gut microbiota in systemic immunity, particularly in IBD-associated inflammation of the eye, skin, liver, and joints. His lab found that in IBD-associated spondyloarthritis, which has no diagnostic biomarkers but is treated with medication that has anti-inflammatory and antimicrobial effects, E. coli bacteria are more prevalent in gut microbiota and Th17 mucosal immunity is increased, suggesting a role for gut microbes in inflammatory joint pain in IBD. The researchers are continuing to study how gut microbiota are involved in inflammation occurring outside the intestine.
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Presentations available from: Jonathan Braun, MD, PhD (University of California, Los Angeles) Stanislav Dusko Ehrlich, PhD (French National Institute for Agricultural Research; King's College London, UK) Sarkis Mazmanian, PhD (California Institute of Technology) Paul W. O'Toole, PhD (University College Cork, Ireland) Noah W. Palm, PhD (Yale University School of Medicine)