Symbiotic Heirarchies of the Intestinal Tract: Implications for Health and Disease Woody Emlen MD

Our Gut Microbiome Woody Emlen MD S ymbiotic H eirarchies of the I ntestinal T ract

Why Now? Hot topic in medical and lay literature (NY Times/Economist/Science/Nature/TED/Mothe r Jones) Explosion of information over past 10 years Significant medical implications beginning to emerge (obesity, diabetes, allergy etc).

What’s the Big Deal: What’s New? Culture – Identify organisms by morphology/chemical tests BUT - Only 20-30% of observable stool organisms grow in culture Past years – Identify by DNA analysis (metagenomics) – 16s rRNA sequencing – Full DNA sequencing Revealed almost unbelievable diversity and numbers!!

Bacterial Culture in Petri Dish

We are not Alone!!

Some Most of my closest Friends are germs! 100,000,000,000,000 bacteria on/in our body – 10 pounds of bacteria!! Several “biota”in multiple sites: – Skin – Mucosa (oral, vaginal) – GI tract (Large intestine>> small intestine) Each biota is a complex ecosystem in itself

Microbiota Sites

Are we really Human? 350,000,000People in US 7,000,000,000People on earth 10,000,000,000,000Cells in our body 100,000,000,000,000Bacteria in and on your body - RIGHT NOW! Are we merely a carrier for a huge bacterial colony?

Terminology Microbiota – the organisms (bacteria, fungi, viruses, etc) living in/on us. Microbiome: - the DNA contained in our microbiota – 23,000genes in the Human genome – >1,000,00genes in the microbiota microbiome

The Gut Microbiota different species – >5000 taxa (identifiably different on DNA analysis) – Most bacteria anaerobic Densest bacterial colony on earth (> /cm 3 ) Extremely complex ecosystem (rain forest)

YUCK!!

Questions Where do they come from? Why are they here? – what’s in it for us / for them? How do we protect ourselves from them? Do they have any effects on our lives – for better or worse? Can we control / manipulate them?

Where do they come from? Sterile gut at birth Initial colonization from mother during childbirth – Significant differences in MB from babies delivered by C-section – Differences based on breast vs formula feeding – Differences based on diet MB grows in complexity over first 3 years – gradually stabilizes to “adult” MB – Development can be significantly altered / limited by early antibiotic use

Effect of Diet on MB Gnobiotic mice fed varying diets J J Faith et al. Science 2011;333: Published by AAAS

Microbiota Stability “Adult” MB relatively stable over time – Stable by function rather than by specific organisms (stability is with microbiome) – Shaped by diet, host genetics, family members, family pets, family behaviors May be disrupted by antibiotics / stress / disease / drug / infection / etc – Return to baseline MB over 2-4 weeks in most individuals but may develop new stable MB – Decreased complexity of MB with age

Why are they here? If you can’t fight ‘em, join ‘em!! Advantages to both: co-evolution has led to true symbiosis – Microbiota: Nice warm, anaerobic home Constant food supply (50 tons!) Surrounded by friends – the good life! – Host: Supplement nutrition Remove toxins Protect from pathogenic bugs Enhance / augment immune system

The Gut Microbiota: Function Express times more genes than the host (us)!! Digestive enzymes Products of fermentation signaling molecules vitamins – Metabolism/Nutrition Extract nutrients and energy from our diet Breakdown of xenobiotics (including drugs) Signals to liver/fat cells to modulate metabolism – Resistance to pathogenic bacteria – Shape immune function & repertoire

Nutrition Enhance absorption of calories/nutrients – Gnobiotic mice require 30% more calorie intake – Break down non-digestible polysaccharides – Generation of short-chain fatty acids (<6 C; promote colon health, anti-inflammatory in colon) Convert / generate some essential nutrients to usable/absorbable forms – Vitamin K and B, folic acid derivatives – Generate small molecule “messengers” (36% of small molecules in circulation are MB origin) Alter Drug Metabolism – Drug breakdown

Gut Microbiota: “Enterotypes” Several general “types” of ecosystems – Characterization very complex and still inexact controversial Best defined by functional activity rather than species types or heterogeneity More species diversity is usually a more stable and “healthy” microbiota. Useful in seeking correlations with health status/disease

MB, Obesity and Diabetes MB of lean and obese mice differ “Obese” MB is more efficient at extracting energy from diet Obesity and diabetes are transmissible to gnobiotic mice with appropriate MB colonization non-obese MB Obese MB Differences in MB of obese and non-obese humans similar to differences in mice

MB, Obesity and Diabetes in Humans Compared MB of lean and obese twins (monozygotic and dizygotic) and their mothers. – MB similarity between pairs of monozygotic and pairs of dizygotic twin equal; both very similar to their mothers. MB from lean twins similar to each other but different from MB of obese twins. CONCLUSIONS Environment is major determinant of MB rather than genetics May be able to define/describe “obese” MB Experiments in humans underway in Netherlands (at 6 weeks – improved insulin sensitivity but no weight loss)

Can MB Generate Harmful Molecules? Red meat/high fat diet accepted as a risk factor for CVD – Rich in lipids / also rich in choline & lecithin Metabolite of choline (TMAO) induces heart disease in mice Is TMAO a risk factor for CVD in humans? – YES – Epidemiology study of 4000 subjects – high TMAO gave 5 fold increased risk of heart disease (after correction for other risk factors)

Where does TMAO come from? Choline challenge given and TMAO levels measured (visit 1) Treated with 1 week of antibiotics to remove MB –challenge repeated (visit 2) Third challenge given 2 weeks after antibiotics stopped after MB recovery Normal After antibiotics Recovered from Abx

Dietary red meat / Eggs / cheese Conclusion: Foods thought to cause / contribute to heart disease exert their effects through the action of the gut Microbiota.

The Gut: Immune System Interactions Priority ONE – protect me from the bugs!! – 1 cell thick layer separating us from bugs – Need to protect surface area as large as a tennis court – Leakage of bacteria into blood – sepsis & death When Pathogens invade: – Produce toxins that kill competition – Induce body to produce endogenous toxins to which they are resistant (decrease competition) – Induce inflammation – Similarity to antibiotic effects – markedly damage the normal MB

Pathogen Resistance by MB Competition for nutrients (stable ecosystem is a complex ecosystem) Production of anti-microbial peptides (direct and induced) Mucous layer to keep bacteria away from gut wall Drive Immune System to produce antibodies (IgA) and immune cells Disrupted by antibiotics

Clostridium Difficile Antibiotic treatment leads to overgrowth of C. Difficile – chronic diarrhea Treatment with antibiotics to try to kill the C Difficile – requires repeated treatment Fecal Transplants to restore normal MB in patients unresponsive to antibiotics – Given by enema / colonoscopy / NG tube – 91% success rate at 6 weeks

Our Gut Ecosystem restores Restores gut ecosystem

Effect of Gut MB on host Immune System Drive development of gut immune system – Gnobiotic mice Shape repertoire of immune responses – What organisms do we recognize / respond to? Maintains “balance” between different types of immune response (TH1 vs. TH2)

Allergy/Asthma Hygiene Hypothesis Increasing incidence of allergy/asthma in western / “1 st world” countries “Civilization” of East Germany Excessive “hygiene” pre-disposes our children to develop allergies/asthma Microbiota: – Childhood antibiotic use associated with increased allergy/asthma – Mouse models – early antibiotics: more severe asthma

Types of Immune Response Type 1 Kill internal pathogens Internal inflammation Cell/bug death Type 2 Remove external irritants Sneezing Coughing Itching Excess Inflammation Autoimmunity Allergy Asthma Microbiota

Overview: MB Effects on Disease Host Genetics Gut Microbiome “Type x” Disease Early Environmental Effects – maternal /other Genetic Predisposition Epi-genetic effects of MB products Generate / deplete Metabolites Environmental Exposures Diet

Gut Microbiota: Treatment Can we control or modify our microbiota to treat / prevent disease or alter our disease risk? – Fecal transplants – Probiotics – Prebiotics

Probiotics Strains of microorganisms which confer health benefits on the host – A “probiotic” MB is one that supports health – A “dysbiotic” MB is associated with disease Probiotics as supplements must: – Survive and metabolize in the gut – Confer beneficial effects – Non-pathogenic and non-toxic*

Probiotics – Current Status Clinical Studies – few controlled – Decreases antibiotic induced-diarrhea 50-60% – No benefit in travellers diarrhea – ? Effect in Inflammatory Bowel Disease – No effect in diabetes/obesity (to date) – Generally safe (few cases of sepsis) Shotgun approach: – What is the appropriate probiotic or prebiotic for any given disease not yet defined – MB of individuals not defined – unclear what is needed

Probiotics – Current Status $16B industry Strain / number of organisms per dose varies from 5-100B Storage conditions / QC vary Survival of organisms in gut and change in MB as yet undocumented Tremendous therapeutic potential in future Buyer beware at present

Prebiotics Foods that promote growth and activity of beneficial bacteria – Resistant to digestion (acid/enzymes/absorption) – Able to be fermented/metabolized by intestinal organisms – Promote growth/activity of beneficial intestinal organisms – Egs: Fiber/complex polysaccharides Still very little research on what foods/prebiotics promote what bacteria, and in turn what bacteria we want to promote Combined with probiotics - synbiotic

Our Gut Ecosystem

The Future Human Microbiome Project (HMP) underway Probiotic bandwagon is well underway – Right now a better financial investment than a health investment – Not yet a clear-cut good health investment – but it likely will be In 20 years: – We will all know our “fecotype” just like we know our blood type – Personalized medicine will include and utilize knowledge of both our genome and our microbiome.

The Future SHITTY treatment from our doctor may be just what we all want and need!!