The Human Microbiome: A New Frontier in Health

(upbeat music) - I'm excited to be with you tonight to discuss a relatively new and I think incredibly exciting area of ​​research that is really changing our view of

human

biology. The field of

human

microbiome

research. And that really describes the study of the intensely diverse communities of fungi and bacterial viruses that live in and on the human body and understanding how they shape our

health

. So what are we going to discuss tonight? Well, first things first. How do you study entire microbial communities? What tools do we have and how do we understand not only who is there, but what they do and how they interact with the human host?

And we are experiencing a rapid whirlwind of the human

microbiome

. Human Microbiome 101. And then I want to move on to some of the work we've been doing to use the very early gut microbiome to understand respiratory diseases that occur years later in childhood. So it's not the typical thinking in the box on this one. And how we can use the microbiome not only to predict the development of allergic asthma, but also to understand why it develops in these children at a very young age and develop new therapies to intervene early to prevent the development of diseases.

And then I want to give you a brief overview of what's next. What's in the future? What is the crystal ball for this field? What are we developing here at UCSF and beyond to use the findings in this area to really develop what we see as a new field of microbiome medicine? That's why when I start my readings, I like to start at the beginning. The beginning: the birth of the planets. And point out that the first and most successful organisms on Earth are microbes. They are bacteria. They have been around longer. They are the most successful.

They are also the most numerous, diverse and ubiquitous. Everywhere we look on this planet we can find microbes. They have evolved and adapted to live in the most extreme environments. It could be anything from an acid mine to incredible pressure at the bottom of the ocean to incredible extreme heat and chemical exposure, for example. So they are a depth. They can live in the most extreme environments. And like I said, they are everywhere. So we and every other biological entity on this planet have evolved in a microbial soup. And in fact, we not only evolved in a microbial soup, but we also co-evolved with microbes.

They live inside and outside of us and, in fact, we depend on them for functions that we ourselves do not encode in the human genome. But how do we study microbiology? Well, traditionally we have had a very reductionist view. What we have traditionally done is take microbes from their environment, grow them ourselves in laboratory media under match conditions and study what they do. And nothing could be further from the truth about how these microbes exist. They are actually quite social. They live in diverse communities like us. They communicate with each other, using small molecules to sense who is near them and respond to microbes that are near them.

So how do we get to these organisms, many of which we've never grown? We don't know how to raise them, we don't know what they eat, we don't know what they live on. We move on to molecular tools. So the kind of workhorse in the field of microbiome research is using DNA-based methods to identify microbes without having to culture them or isolate them from a sample. We take a sample, extract the DNA, and remember that the DNA comes from every microbial cell in that sample. There can be different types of microbes and one approach we have is to target specific genes like this one called the 16S ribosomal RNA gene.

This is a gene found only in bacteria. It's not found in higher organisms and it's a great biomarker gene to identify which bacteria it comes from because it has these regions in the gene that are actually highly conserved in all known bacteria. We then use those highly conserved regions to anchor an assay that we essentially need to make copies of the intervening region. And the regions between those really conserved regions of the gene are what we call hypervariables. There the order varies. And it varies depending on the bacteria from which the gene comes. So we can make many copies of these genes and then sequence the hypervariable region to find out who it came from.

And that way, as if it were a fingerprint, we can generate what bacteria there are and how many of each bacteria. What is the relative abundance? And this is very useful for comparing very large cohorts of samples where we just want to know who is there and how they differ, for example, in

health

and disease. We have a similar tool for looking at mold. There are many different regions that we can look at with the same type of technique, but we tend to use this so-called gap two region. Again, we amplify that part of the fungal genome, we sequence it and then we can see what fungus it actually evolved from and that way we look at the fungal communities and how they are composed.

Who is in that mushroom community. But microbiome assessment tools have evolved rapidly and expanded in capacity in recent years. And now, instead of just looking at one biomarker gene, we can take all the DNA that we've extracted and sequence all that DNA and then put those pieces back together, essentially recreating the genomes of all the microbes in that sample. compile. And this is no small feat, as you can imagine. I joke, although it's not really a joke, and say it's like handing you Tolstoy's "War and Peace" in pieces and asking you to put it all back together in a readable format.

That's the computing power we need. It's huge to do this work, but it's something we've developed very quickly over the last few years. So while biomarker sequencing tells us who is there, shotgun metagenomics tells us the genes these organisms encode and what they can do. But we have taken this field even further. We can also extract RNA from a sample. It is a different type of nucleic acid. And it is actually the transcription of those genes. So it's what the microbial community actually transcribes from their genomes. Wow, they are responding to the current circumstances. And we can sequence those groups of extracted RNA also through sequencing and we call this metatranscriptomics.

It gives us a snapshot of the genes expressed at the time of sample collection by organisms in the microbiome. But what's even more exciting is that we can look even deeper. These are all next-generation sequencing-based tools for looking at microbiomes. We can also use mass spectrometry, the ability to identify small molecules. And we can use this to look at the sets of proteins produced by the microbiome to understand the proteins they produce, including all the enzymes and catalytic functions of the microbiome. But what interests me most is metabolomics. Looking at small molecules. Remember, I said this is how microbes communicate with each other.

Basically, this is how cells communicate with each other, regardless of their microbial or host state. And this to me is the lexicon that governs the interaction between the microbial host and the human host and this is where we think about the next

frontier

and we all already realize that the next

frontier

really lies in this area. So the application of these tools, and particularly DNA-based tools, has greatly expanded our view of bacterial life on this planet. This is the tree of life. This is all. We're in one of the little branches down there with the eukaryotes.

These at the top are all bacteria. And this is a study that was published in 2016. Everything in purple is new bacteria that were only identified in this study using molecular methods. We have thus greatly diversified the bacterial tree of life and we suspect that this also applies to viruses and fungi. We just need to catch up on developing tools for those kingdoms of microbial life. But what these tools have told us is that there is a much broader range of fungi and viruses. Especially those that exist in the human body than we were previously led to believe based on cultural approaches.

And all told, the application of all these tools to interrogate the microbiome has made us realize that we are not alone. In fact, we are superorganisms. We are a conglomerate of microbial and mammalian cells that have coevolved over time and are colonized by microbes inside and out. It is simply about observing the microbial diversity on the surface of the skin. Everything in red is some type of higher diversity, in blue are areas of lower microbial diversity. This involves observing the molecules produced by these microbes on the skin with mass spectrometry. And here you can see that even where there are regions where there are not as many microbes present, there is an enormous biochemical diversity of molecules that are produced in those places.

Produced by microbes, produced by host cells in response to microbes. A rich molecular lexicon is found in these sites. That's just the skin. And those are actually considered sites with very low microbial load. We harbor the greatest load and diversity of microbes in our intestines. Especially in the distal intestine. And these microbes are not just spectators. These microbes influence how our gastrointestinal cells behave and function and respond to this microbial zoo in the lower intestine. And I will say that it is not just the intestine. There are clearly microbes in the mouth and throughout the entire gastrointestinal tract.

They differentiate at several locations along the gastrointestinal tract. And we think that's because the prevailing conditions are different. If you think about the stomach, the pH is very low. There is very little oxygen in the lower part of the gastrointestinal tract. These are strong selective pressures that drive the types of organisms that like to thrive in these various niches along the gastrointestinal tract. But I think it's really amazing to think about the extent to which our microbiome dwarfs our human genome in terms of genetic capacity and the genes it encodes. This is a study of European, Asian and American populations.

Just over 1,200 fecal samples were sampled and examined using shotgun metagenomics. So looking at all the microbes and all the genes encoded by the microbiome in those 1200 samples. And the surprising thing is that almost 10 million microbial genes were found in those approximately 1,200 individual fecal samples. I want you to think about that for a moment. That is incredible. This is an additional microbial genome that we carry with us. These genes are not silent. They express themselves actively. And we depend on these genes for things like digesting the components of our food, digesting our medications, and informing and influencing our immune response.

So these are an important part of our physiology. An important part of what makes us healthy or sick. And to add the complexity that this is not just one type of microbiome that we have at one site. We develop our microbiome at a young age. We are born with the very simple microbiome that we inherit from our mothers. It occurs in the womb or is contributed to by the birth process. Babies born through the vaginal canal have a predominance of lactobacillus species, the dominant organisms in the female vaginal canal. Babies who reach sunroof via C-section often end up with organisms on their skin.

Staphylococcus and streptococcus. This suggests that postnatal exposure at a very young age influences the microbial communities found in the early stages of the gut. And as we progress through early life development, we now know that a whole range of factors influence and shape the types of microbes and activities of the gut microbiome. Things like diet in early life and exposure to antimicrobials, as I mentioned, have had a strong influence on the types of microbes that exist and how they function. We continue to expand the diversity of bacteria we have in the intestine for about three years.

At that time, the diversity resembles that of an adult, but the functional genes in that microbiome at three years old are very different from those of a healthy adult. Throughout life, we continue to shape our microbiomes. In fact, as an adult, I think of them as a story of your exposures in life. Things like drugs, diet, infections, sex hormones, and even toxins in the environment can act as strong selective pressures on the microbes present and what they produce and therefore how they interact with the host. And to really amplify this and increase the complexity, if we take a moment in time, not all microbiomes are the same.

In this case, it is a study of the gut microbiome in developed and developing countries in adults. Here, in red and green, we have gut microbiomes from Malawian and Amerindian populations. In blue we have the US population. And inIn reality, each point is a profile of what type of bacteria were in the intestinal microbiome of these individuals and how we work with this immense amount of data that we are generating. We asked to what extent microbiome profile A is comparable to all other microbiome profiles in our cohort. And we calculate a distance. How similar it is, how close it is in terms of what microbes are there and what relative abundance, how many of them there are.

And that's just a visualization of this distance calculation. So if we have two points representing two gut microbiomes of individuals in these studies plotted near each other, that means those two gut microbiomes are very similar. But what can be clearly seen here is that the microbiomes of the American gut are very different from those of the Marindian and Malawian populations. And although the populations of Malawi and Merindia are located on two different continents, their gut microbiomes in these less developed countries are more similar to each other than to the gut microbiome of the US population. To expand on what we have done with our microbiomes in the US, we simply looked at the number of types of bacteria detected in these populations.

We have greatly reduced the breadth and number of different types of bacteria in the US population compared to less developed countries. And the really cool thing about this study is that we have clues as to why this might be happening. When the study looked at shotgun metagenomics looking at all the genes, pathways, and these microbiomes, what really differed between these populations, what really stood out was that the adult population of Amerindians and Malawians were really enriched in alpha-amylases. This is an enzyme that breaks down complex plant polysaccharides. Therefore, the diet in Malawi and the Merindian population is mainly based on plant polysaccharides, a plant-based diet.

In the US population we see tremendous enrichment of microbial metabolic pathways to process simple sugars. It is found in processed foods, as we all know. So at least one characteristic, something we know, is driving these differences in the gut microbiome of these populations: dietary differences in what we consume. And this was recently reinforced by Pete Turnbull, a faculty member here. This was a really excellent study based on the diet of 10 people. And what Peter did was take those people and look at how a plant-based or animal-based diet can really impact a healthy gut microbiome. And here we'll just show you the amount of fiber in the diet of these individuals before we started the study, then they were fed a plant-based diet for four days, the other participants were fed an animal-based diet for four days, so Whereas one would expect the fiber content in a plant diet or the plant polysaccharide content to be quite high, in an animal diet it is very low.

Additionally, fat intake is lower in the plant-based diet compared to the animal diet and the protein content is also dramatically different between these two diets and you could see that these key nutritional components really change with the introduction of a plant. diet or animal diet. What really stood out was that when Peter recalculated the distance, how similar individuals' microbiomes are after starting their plant-based diet compared to before starting their plant-based diet, he didn't really see many changes. . Plant-based diets don't really alter the gut microbiome. In comparison, the introduction of animal diets has actually increased this distance.

And what that tells us is that the microbiome is very different from the microbiome that existed before the introduction of the plant-based diet. But the important thing is not only to change the composition of the microbiome. What the study also showed is that the molecular production of the microbiome is changed by changing the diet. And here we can see that two important short-chain fatty acids, acetate and butyrate, were significantly reduced in the animal-based diet compared to the plant-based diet. And that makes sense, because these are products of microbial fermentation of plants, of fibers. And what is really crucial is that these short chain fatty acids are crucial energy sources for the cells of the intestine, they are antiproliferative and anti-inflammatory.

And we think they have these activities because we've co-evolved with these microbes that have traditionally fermented our plant foods into these little molecules that quench inflammation and promote some kind of health in the system. Based on this, I'm sure you won't be surprised that we are discovering that an increasing number of diseases are linked to alterations of the microbiome. And things like the skin microbiome are altered in dermatological conditions like psoriasis. But what's really interesting is that we now say that conditions such as obesity are also related to the alteration of the intestinal microbiome. But what's most exciting to me is that we're finding that conditions that are very difficult to treat and are really out of our control, like depression and autism spectrum disorder, are also linked to alterations in the gut microbiome.

This suggests that the gut microbiome may actually influence distant organs. And there are some really important groundbreaking studies that have shown this. They have shown that alterations in the gut microbiome are related to autism spectrum disorder and also cardiovascular diseases. But the important thing that these studies have shown is that it is the microbial metabolites, the microbial products, that are responsible for these conditions and much of this work has been done in mice with some follow-up work in humans. This suggests that the gut is not like Las Vegas: what happens in the gut doesn't stay in the gut.

It actually gets into the bloodstream and into these little molecules and maybe there are some clues that even the microbes themselves can move to other places in the body and change the physiology of the organs there, contributing to the health or disease of those remote organs, but as? Do we really know? Is the gut microbiome responsible for this? Well, that evidence comes from some really fancy mouse studies. So in these studies, in this case that I show you, the feces of obese individuals and lean individuals became germ-free mice. These are mice that do not have any existing microbiome.

They were bred to lack a microbiome. They are not particularly healthy mice, but they were bred to have no microbiome. They are a wonderful vessel to study how microbial introduction into a pristine type of environment can influence host physiology. And that's what these studies have shown us. Transferring the obese microbiome to a germ-free mouse creates an obese microbiome in that animal, and those animals gain weight at a much faster rate than animals that receive the lean microbiome. It suggests that the patient's disease phenotype can be transferred by transferring that patient's gut microbiome into a mouse. That's pretty incredible.

This means that the microbiome in this case is largely responsible for obesity. The interesting thing is that it is not just about obesity. This has also been demonstrated in the case of Kwashiorkor. It is a devastating disease with neurological deficits that are quite common in underdeveloped countries like Bangladesh. The same. Transfer of Kwashiorkor's gut microbiome or feces to germ-free mice causes a debilitating disease in those animals. More recently, it has even been shown to be used for autism spectrum disorder. Feces from ASD patients transferred to germ-free mice induce neurobehaviors consistent with the symptomatology of the disease.

Again, we found multiple disease markers where we can recapitulate the characteristics of the disease in a mouse that receives the microbiome of patients with the condition. So what can we do? Well, we call it yellow soup for the soul. Fecal microbial transplant. I'm sure you've all heard of him. It is not new. I call it yellow soup for the soul because in 5th century Chinese medicine there are records of how yellow soup was produced from feces as a treatment for gastrointestinal disorders. We recently rediscovered fecal microbial transplantation. And what's really exciting is that it essentially does what we do in mice, but instead transfers the healthy gut microbiome of a healthy donor to the gut microbiome of a patient with a disease, condition or infection to try to change the microbiome. intestinal to rebuild. of the patient and treat the disease.

Therefore, it is 92% effective in patients with a Clostridium difficile infection. Antimicrobial treatment for Clostridium difficile is approximately 30% effective. And in this study that used fecal microbe transplantation to treat Clostridium difficile infection, they actually stopped the study early because it actually wasn't, they couldn't treat patients with this treatment because they saw 92% efficacy. compared to 30% with vancomycin. . patients treated with microbes and cones. It was unethical to continue the trial and not use it to treat patients. And we actually offer this at UCSF as a treatment for Clostridium difficile infection. It has also been used in a small early pilot study of children with autism spectrum disorder.

There are 18 or 19 children in the study. There they saw a significant reduction in neurobehavioral symptomatology in these children. In this case, instead of a single treatment, a colonoscopic administration of the fecal suspension to the diseased intestine is performed. They did that initially, but then they followed a month of sustained microbial pressure in which the children consumed freeze-dried fecal capsules and that was enough, one month of treatment, to significantly reduce the neurobehavioral deficits in these patients. They also recently followed up with these children two years later and this effect persists. And in fact, they have seen even greater improvements in this small group that has been treated.

So this is now being investigated clinically in a much larger nationwide placebo-controlled trial as a possible treatment for autism spectrum disorder. At UCSF we look at inflammatory bowel disease. I'm in the gastroenterology department. I can tell you that we have looked at Crohn's disease and ulcerative colitis and fecal microbial transplant does not work for Crohn's disease. At least as we tried with a single colonoscopy. We had several side effects and stopped the study. And I think it's important that that message gets across as much as the message of 92% efficacy of Clostridium difficile. It is not the same.

The microbiome is not the answer to all of our patients' conditions. And I think it's important that we be careful and that we be thoughtful about how we implement this field and how we use it to treat our patients. Using a very similar approach to the study of patients with autism spectrum disorder, we now have a response rate of approximately 40% in our patients with ulcerative colitis. And that's really exciting. You know our biologics are about 20% effective and we test different biologics on patients to see which one works for them. But we see a 40% response rate with fecal microbial transplant.

We have ideas on how to improve this even further and I'll talk about some more later in the study. For me we are at a turning point in human biology. We just discovered that we have this extra microbial genome that really influences our health, determines how our cells function and affects our state of health. And what I want to change now is talk about how we use this field to address a disease that I know, probably everyone in the room knows someone with asthma. Currently in the US, approximately 11% of our population has been diagnosed with the disease and is growing.

And you can see on this map that it is a disease of Westernized countries. This is a disease of lifestyle and environment. It is not necessarily a genetic disease in the typical sense of the word. What I find most alarming is that the prevalence of this disease has increased most dramatically in the pediatric population. Children are disproportionately affected by allergic asthma, the predominant form of asthma in this country. And for those who are less familiar with the disease, it is characterized by a fairly specific immune dysfunction. Children with allergic asthma have many fewer specific types of T cells called regulatory T cells.

They produce this molecule called IL-10. And you have to think that these cells inhibit inflammation. We need them to reduce inflammation. So children with asthma have many fewer of these types of cells and, on the other hand, they have many more. TheseThey are another type of T cells called T2 cells. And they produce three other molecules called IL-4, IL-5, IL-13 and increase inflammation. These children are also characterized by having very high concentrations of this IgE antibody in their bloodstream. So they are the cardinal dysfunctional immune system characteristics of allergic asthma. Something to remember as we move through the rest of the presentation.

I think what also surprises me is that while we can treat our patients with corticosteroids, long-acting beta agonists, we don't have a cure. And that's what really drove me into this field and I started thinking about early life and what are the factors that influence disease development and how the microbiome could be the canary in the coal mine for development. asthma and allergies in childhood. And what attracted me to that idea was really the opportunity to stand on the shoulders of giants. There are many studies that have attempted to determine the onset or origins of the development of allergy and asthma.

That's why there have been a lot of very large studies, birth cohort studies where babies are followed from birth to childhood. You know their early exposure and know if they developed allergies or asthma years later in childhood. And these studies are really consistent in the factors that we know increase the risk of disease. There are things like formula feeding, antimicrobial administration, and C-section. And if those factors sound familiar, they are among the things I told you at the beginning of this presentation that determine the composition and activities of the gut microbiome. On the other hand, a reduced risk of allergies and asthma in childhood is associated with breastfeeding, exposure to livestock and animals.

And in fact, we have shown it to cats, mice and cockroaches at the center. All are vectors of microbes and increase microbial diversity and microbial exposure of babies at a very young age. And we think that's important because we think that the baby's environment serves as a library of microbes available for accumulation in the gut microbiome and elsewhere as we develop our microbiome in that critical period of the first years of life. But before we really got into this area, we were still interested in asking questions in models. Can the gut microbiome really influence the respiratory system?

Because no one had really proven it. And to do this, we did a fairly simple study in which we took mice and fed them daily with this species of lactobacillus. We did this for a week before sensitizing the animals' airways with cockroach antigens. IT stands for intratracheal and CRA is cockroach antigen. We then exposed the airways of these mice to an antigen that causes allergic inflammation. As a control group we had animals that did not receive Lactobacillus johnsonii. And what we found was that in the animals that received Lactobacillus johnsonii, you can see that we significantly reduced IL-4, IL-5, and IL-13, the three molecules that I told you make up the group of cells.

They produce T cells that promote allergic inflammation. And this was true whether we looked at the expression of these genes or the protein of these genes. And what was even more compelling is what the airways of these animals are like. These are the animals whose airways we have sensitized and who do not have lactobacilli in their intestines. These are the respiratory tracts of those who received lactobacillus supplementation. The air spaces of these animals are absolutely pink and closed with mucin. That pink spot stains the mucin. Then they are completely filled with mucin. These are severely inflamed airways and this does not occur in animals that have received oral supplements of lactobacilli.

We started thinking about this. Is it just allergy or is it really a deeper protection of the airways that occurs when we change the microbiome by introducing microbes into the gut? So we asked the same question, but we didn't use any allergens here. Now we use respiratory syncytial virus or RSV. We consider it an asthmatic virus. Children who have an RSV infection in the first months of life that requires hospitalization are significantly more likely to develop asthma. It is a kind of warning sign for asthma. And then we have this model where we have used live Lactobacillus johnsonii or heat-killed Lactobacillus.

Do we need a metabolically active microbe to provide protection to these animals? And PBS is simply saline solution. That is the control in this study. And we know that when we infect these mice with RSV, they have this kind of very predictable infection dynamics and by day eight we can see profound pathology in the airways. So what we found is that when we tested the airways of these animals for responsiveness, only the animals that received live Lactobacillus johnsonii had significantly reduced reactive airways. And they were also the only animals that had significant reductions in allergic inflammatory markers and molecules in their airways.

Again, IL-4, IL-5 and IL-13. This told us that we needed a live microbe to confer protection to the airways. Which is what this is pointing out. But we started to think about how this happens. What actually happens before we get to that stage where we can see differences in airway pathology? So we moved back the timeline in this model and just asked with the same type of model, now we're just looking at animals supplemented with live Lactobacillus johnsonii versus PBS, what happens on the second day. And we were particularly interested in whether there were metabolic changes in these animals.

Whether small molecules produced by an altered gut microbiome could hold the secret to the response to viral infection. And I don't expect you to read all of this. This is where we use that mass spectrometry to look at all the small molecules that are produced in these animals in their serum, in their circulation. This is what we see in control animals two days after we infect them with respiratory syncytial virus. Everything in blue has gone down from the baseline, everything in red has gone up. There's not a lot going on. And I think you'll agree that that's true when I show you what happens in animals that receive live Lactobacillus johnsonii.

Now, two days after the viral infection, we see in the respiratory tract this immense capacity to produce a whole range of amino acids, peptides, but in particular lipids, and when we saw this list of lipids, we got very excited. Because in this list of lipids there is a wide range of things like polyunsaturated fatty acids that we know reduce inflammation. And that suggested to us that when we alter the gut microbiome of these mice, we change the metabolic output of not only what's in the gut but also what's in the circulation of these animals and that's what leads to protection against viral infection in the respiratory tract.

But we wanted a little more evidence of this. So we did one more experiment. We take what we call bone marrow-derived dendritic cells. These are immune cells that are really critical in response to a viral infection. And we incubated those immune cells with the blood, the plasma of animals that received the control PBS and were subsequently infected or those that had Lactobacillus johnsonii introduced into the intestine and were then infected. So those who had that two-day lipid attack that we saw. And then we take those dendritic cells, those immune cells, and we ask them how they now respond to the virus when they encounter it.

Could the products we see in circulation change the activity of immune cells? And that's what we found. The immune cells that we know were incubated with the plasma of the animals that had Lactobacillus johnsonii are now significantly less inflammatory and significantly less activated and have a significantly reduced ability to present antigens to participate in an inflammatory response. So what this tells us is that by changing the gut microbiome we can change the metabolic output of the intestinal system and we can actually protect the airways of those animals. And it seems that part of this is due to the production of these metabolites.

What I didn't tell you is that we began to observe some of those metabolites and found that one of those polyunsaturated fatty acids referred to this phenotype in the cells. So we were right to think that those anti-inflammatory lipids are perhaps what's driving this change in how our immune cells function in this mouse model. That's all on mice. This is incredible. But that is a model system. Could the early-life gut microbiome be altered in babies who develop allergies and asthma? And perhaps beyond a perturbation about who is there, could the metabolites produced by the gut microbiome in the first years of life actually be the key to differentially priming immune cells in children, in babies who later Will they be children with asthma?

And so we really wanted to base this on something that was very solid. And so we think about microbiome development in the early stages of life in the same way that any other ecosystem develops. And we've studied ecosystem development for a couple hundred years. So they are a pretty good framework. We know how ecosystems develop. And one of the things we know about the development of ecosystems is that the first colonizers, the first species in a previously pristine ecosystem, can actually shape the conditions in that ecosystem and species accumulate trajectories over time. So what this suggested to us is that maybe there are different types of seed microbiomes.

Different types of microbiomes in early life leading to different trajectories of microbiome development. And remember, the microbiome educates the immune response. And we think that could lead to different immune maturation and lead to the development of health or asthma and allergies years later in childhood. And again, as I mentioned, we started thinking about how this could work. And we started thinking about what we've seen in the field of neurology, the gut microbiome, or the field of cardiology: that gut microbial metabolites can shape the behavior of remote organs. And we've seen it in our mice, so we think it's not just a disrupted gut microbiome in the first few years of life, but maybe it's the molecules that that microbiome is producing really skewed immune development in those babies.

So one of the first studies where we addressed this was a study of the gut microbiome of healthy babies at high risk for asthma. And they are considered babies at high risk for asthma because they have at least one parent who has asthma and this is the meconium microbiome. This is the first bowel movement of newborn babies. These forms in the womb. And here I show you another one of those graphs where it is one of those distance graphs. In red are high-risk babies, in green are healthy babies. And you can see that they are segregated along this axis.

They are spatially separated. In reality, they are significantly different. Babies at high risk for asthma begin life with a very different microbiome than healthy babies. And what's exciting and consistent with what ecosystem theory would predict is that those babies follow a different trajectory of microbiome development. These are healthy babies and accumulate bacterial diversity at a fairly rapid rate during the first year of life. In contrast, the high risk of asthma and allergies in babies has delayed the diversification of their gut microbiome. And remember, each species of microbe brings with it its own genome and its own repertoire of genes to the gut microbiome.

So these babies will have a functionally different gut microbiome. They simply do not have the same microbial capacity that a healthy gut microbiome does. But that is simply a study. Can we see this in a population? These are high risk controls versus healthy controls. This is the extreme. Can we really detect this only in a population of babies? And to do this we study a large birth cohort. Again, this is one of those studies where you collect samples early in life and you follow the babies throughout life and we know if, in this case, they developed allergy at two years of age or asthma at four years old. and the part of the study that I am going to tell you about, we had 130 one-month-old babies from whom we took fecal samples.

So we profiled their microbiota using the gene-based approach that I told you about at the beginning, to find out what bacteria and what fungi were present in the microbiomes of these 130 babies. And then it's a pretty large amount of data. At this stage we stop intervening. YouWe asked an algorithm: can you find significantly different gut microbiomes among these 130 babies? And the answer was three. The answer always seems to be three. Again I show you one of these distance graphs. And here you can see what the algorithm called the three different gut microbiomes, we've labeled them neonatal gut microbiome one, two, and three.

They are shown in blue, green and red and are again spatially segregated. They are significantly different in their composition and in fact they are called neonatal gut microbiome one, two and three. These classifications explain around 9% of the microbiota variants that we see in these 130 babies. But the key question is whether starting life at one month of age with one of these gut microbiomes is related to the clinical outcomes we see at two and four years of age. And the answer was a resounding yes. Babies with the NGM3 gut microbiome at one month of age had a significantly higher risk of developing atopic allergies at two years of age and asthma years later, at four years of age, about three times more likely to develop these diseases.

What was different about these gut microbiomes? Well, we found that it wasn't just a loss of bacteria that we saw in the gut microbiome of the high-risk NGM3 baby. We also saw that they were greatly increased for what we consider allergenic fungi, rodatorola and Candida. So it's not just a loss of bacteria, it's also an increase of fungi in the gut microbiome of these babies. And using mass spectrometry, we asked if the metabolic output of this gut microbiome is different. And we discovered that it is and I'm not going to show you another one of those crazy plots where you can't read anything but I'm going to summarize and tell you, just as we saw in our mice, the babies who continued to develop allergies and asthma had significantly reduced acids. polyunsaturated fatty acids, among many other lipids.

And they also increased a lot for this lipid, 12,13 DiHOME, a dietary oxyfatty acid. So what all of this together suggests to us is that NGM microbiomes one and two are actually tolerogenic. They could be educating the immune response in a very different way than the NGM3 microbiome in the intestine, which is full of potential pathogens and metabolically very altered. But how do we test this? We really need to think outside the box. All we had was these babies' feces. Nothing else. So what we thought is that we could take immune cells from healthy adult donors and specifically take the immune cells that govern the allergic response, dendritic cells that present antigen to the T cells and educate the T cells and dictate what they will be when they mature.

And then we purified these specific populations of cells, remember, from healthy adult donors, and coincubated the dendritic cells with the cell-free products of the gut microbiome from the NGM3 high-risk and NGM1 low-risk babies. To be able to prepare those dendritic cells. We let them sit for a while and then cultured them with the naïve T cells and we were particularly interested in what we would see with the TH2 cells, remember the ones that produce inflammatory cytokines, and the T-reg cells, the ones that mark reducing inflammation. And what we found was that the cells that were coincubated with the NGM3 fecal water from that month-old gut microbiome had much higher amounts of TH2, allergic T cells, they produced more IL-4, and those T cells were significantly higher.

They are less likely to be regulatory T cells. Remember that I talked to you about the cardinal immune characteristics of allergy and asthma at the beginning of the talk; here we can recapitulate them using the fecal products, the gut microbiome products of a one-month-old high-risk gut microbiome. This is years before we diagnose the disease. But we were really interested in asking what are the products in that type of fecal medium that produce this immune dysfunction. And we initially focused on this lipid that I told you about 12,13 DiHOME. Because it kept showing up in all of our analyses.

No matter how we collected the data, we always came back to this molecule. So we asked if this molecule could recapitulate the characteristics of that immune dysfunction that I just showed you and that we produce with fecal water. And what we found was that, critically, this molecule, as its concentration increases, depletes those regulatory T cells and reduces their ability to produce the anti-inflammatory molecule IL-10. Now we have a molecule that appears to actually distort a very critical part of the immune response that we need to reduce allergic inflammation. That's why we wanted to test it on a mouse model.

What we did is the same mouse model that I presented to you earlier, but here, three hours before challenging the airway with a cockroach, we injected this lipid into the intestine of a group of these mice and asked if it exacerbated the allergic response. in the respiratory tracts of these animals. And what we found was a resounding yes. Here are our controlled animals. Pleasant and clean air spaces, here the animals are sensitized. All of these little blackheads are inflammatory cells found around the air space. As you can see, they're constricted, there's pink mucin there. These are the animals that received that lipid in their intestines before we sensitized them.

We have now completely occluded their air spaces with mucin and inflammatory cells. And based on what we've seen in a test tube, these animals have significantly reduced regulatory T cells in their airways and significantly increased IgE, that antibody that we know is associated with allergy and asthma, in their circulation. So just by simply introducing this lipid into these mice, we can exacerbate the allergic inflammation in their airways. And it suggests to us that elevated concentrations of this lipid in the gut microbiome early in life could actually have the same effect on that critical population of immune cells in these babies, reducing their ability to reduce allergic inflammation.

But we wanted to go a little deeper. This molecule is a product of the metabolism of linoleic acid. Linoleic acid is abundant in breast milk and formula. It is a key lipid in nutrition in the early stages of life. What we found in healthy babies is that their fecal microbiomes are highly enriched with this other metabolite DiHOMEgamalinoleic acid, which is a precursor to a whole range of anti-inflammatory products. The high-risk babies we had shown were highly enriched with this lipid. So our hypothesis is that actually the gut microbiome of these babies has the ability to produce 12,13 DiHOME from linoleic acid.

And we know that the final step in making this product is catalyzed by an epoxy hydrolase, a special type of enzyme that converts 12.13 EpHOME to 12.13 DiHOME. So we dumpster dive into the gut microbiome. We went looking for microbial epoxide hydrolase genes. And we simply quantified them in the gut microbiome of babies who became healthy or those who became ectopic or asthmatic years later in childhood. And remember, this is the microbiome of one-month-old dots. And we found that babies who developed diseases were significantly enriched in bacterial genes to produce this lipid. Not only that, they also had a lot more lipids in their stool.

And we went on to functionally test these bacterial genes and found that three of them were able to specifically produce 12,13 DiHOME, this lipid that appears to be so critical in promoting allergic inflammation as we've seen in our studies. And these are species that every baby has. They have them in their meconium microbiome. Every baby has an Enterococcus faecalis, every baby has a proper bifidam bacteria. But we think the difference between health and illness is that babies who have these species with these bacterial genes are the ones who develop allergies and asthma. And we show that this is true using two birth cohorts.

So we showed that for every doubling of the number of epoxide hydrolase genes in the gut microbiome at one month of age, there is a significantly increased risk of developing allergies and asthma years later in childhood. And that's also true for every nanogram increase of that 12,13 DiHOME lipid in the stool of these babies. And that's consistent when we look at a completely different cohort of babies based here in San Francisco, at UCSF. So what this tells us is that we have moved from a perturbation of the gut microbiome to identifying what we think of microbial risk genes.

These genes confer an increased risk of developing allergies and asthma years later in childhood. And thanks to these genes and their products we are beginning to understand why these babies develop diseases. These microbial products actually distort immune function and reduce key immune cells necessary to reduce allergic inflammation. So we have a new model of pathway by which allergy and asthma can develop. It is one in which babies inherit microbes from their mothers and begin to develop their intestinal microbiome. And those who have a specific type of gut microbiome enriched with microbial capacity to produce this 12,13 DiHOME lipid, have reduced T-reg, these key immune cells for reducing inflammation.

We know from our studies with mice that this lipid escapes from the intestine and actually enters the circulation and reaches the airways. And we think it has the same effect there, by reducing these key immune cells in the airways. And that results in a lack of ability to respond to the pathogenic microbes we encounter with every breath. And those babies develop a pathogenic microbiome in the airways over time and that's what leads to the diagnosis of asthma in these children later in life. We know that this different disturbed gut microbiome results in a different trajectory of microbiome development in the gut of these babies.

So what can we do? Well, we have rationally designed a synthetic cocktail of microbes to introduce early in life, the first day, the day of delivery, into babies at high risk for asthma. These microbes encode all the functions that these babies are missing in their initial microbiome. And the idea is that these microbes shape the immune environment around them and that governs the trajectory of microbiome development and will allow for appropriate development of the microbiome in the early stages of life and also change metabolic output. We will redesign the microbiome of these babies to change metabolic output, change the interaction with the immune response and prevent asthma.

This product is currently undergoing clinical safety trials before being used as a treatment, but it's not just about allergies and asthma. We are also conducting studies on the gut microbiome and obesity in early life. Another plague on our nation. And we are finding very interesting and somewhat familiar findings. Here again we found three distinct gut microbiomes in a much larger cohort of babies. More than 400. One of them confers a significantly increased risk of developing overweight and obesity phenotypes in childhood. These babies are more likely to be formula-fed. And what we found is that the products of that gut microbiome change the way the cells that line the gut absorb and release lipids.

In fact, it accelerates that process, so we think that this excess lipid enters the circulation and, if it is not depleted, is deposited in adipose tissue and this could be a mechanism by which these children eventually develop obesity and obesity phenotypes. overweight later in childhood. It also offers the opportunity that, perhaps again, early intervention in these high-risk babies can change the course of their microbiome development and the metabolic output of those communities and change their course of health. And again, it's not just about obesity. Tiffany Scharschmidt, an amazing professor here at UCSF, is showing that this happens in the skin too.

The first microbes that colonize the hair follicle change or influence the immune milieu in that hair follicle and dictate what other organisms can come to the party and co-colonize in that niche. And this offers opportunities to change the microbial interaction with the host to change the course of disease development. We've also been looking at this in terms of upper airway microbiome development and again we've shown that babies with different trajectories of upper airway microbiome development have a higher risk of asthma and there are specific colonization patterns that we've identified in the upper respiratory tract that not only increase the risk of asthma, but also increase the risk of exacerbation in these children.

Again, thinking very differently about this, thinking of early life as an opportunity to redesign the microbiome in a way that changes thehost physiology and alter the trajectory of disease development in these individuals. So what have we learned from this conference? We know that early life is a critical period in which we build our microbiomes. Not only in the intestine, but also in the respiratory tract and other places in the body. And that the types and, more importantly, the genetic capacity of those microbial communities are really fundamental for promoting the health of human beings. We know that there are distinct founding populations of gut microbes early in life that strongly influence immune function and are related to childhood disease outcomes.

So we go back to believing that we have this canary in the coal mine, we have this very early disturbance that leads to the development of a subsequent disease years later, in childhood. And part of that is that the microbes that are there produce specific small molecules that distort immune function. And we think that this occurs in the early stages of postnatal life and then that biased immune function, that inflammatory milieu in the gut, is strongly selecting the types of microbes that are allowed to occupy the niche in the gut and that's why who observe less diversification of these intestinal microbiomes over time in these babies.

And we're very excited because we really believe that this is a new field that is changing the face of human biology and we're thrilled that this year they launched the Benioff Center for Microbiome Medicine here at UCSF. We're excited about what this field can do and I've really shown you a snippet of the background and some of the exciting work being done in the field. Within the center, we are leveraging, for example, the healthy periodontal microbiome to find new microbes and molecules to address periodontal disease. We are looking at how the gut microbiome is related to multiple sclerosis.

Sergio Bernzini and neurology have really strong data linking this disease to the gut microbiome and he is now involved in a fecal microbial transplant study of this population to ask if that can improve symptomatology in his patients. Katey Pollard, who is here at UCSF, is a computer genius and she is the one that allows us to look beyond who is there and look at specific genes that are the differentiator between health and the development of disease in our microbiomes and in our patient populations. And finally, we are delving deeper into our fecal microbe transplant trials to understand how it works, which microbes and molecules matter so that we can build custom synthetic microbial communities that adapt to specific subsets of patients and don't necessarily go into the environment. with the approach of fecal microbial transplantation of blunderbuss.

And we believe that with this we can improve effectiveness in this population. There are other things they were doing. We are taking advantage of microbes to combat them. Because that's what microbes do naturally in their ecosystems. They antagonize each other and we are taking advantage of this knowledge. We are using fagers. Fagers are like viruses that bacteria have and they are very specific in their target and we wondered if we could use phage therapy instead of antimicrobial therapy. Can we get really specific and go after the key pathogenic organisms that we think are causing the pathology in our patients?

In our upper respiratory studies, for example, we have a couple of very key target organisms: Moraxella catarrhalis. We've seen an emergence in multiple different studies and now we're going to address it with phage therapy to ask if we can specifically eliminate that organism and redesign the microbiome of individuals who have these colonization patterns associated with their disease. There are efforts, not necessarily at UCSF but elsewhere, to harness microbes to express a specific cargo in the gut to produce the molecule IL-10, for example, which reduces inflammation. We are working very hard at UCSF to understand diet and the microbiome.

I showed them a snippet of work from the Turnbull lab. We are using the diet in a pilot study in patients with ulcerative colitis to ask if we can change the microbiome of those patients and induce remission through diet. As I mentioned, we are also developing custom synthetic microbial cocktails and ultimately I think it is the combination of diet and microbiome or specific substrates and symbiotic microbiomes that I think will be most effective in our patient populations. And ultimately, our goal at the center is to focus on the early-life microbiome as an opportunity to intervene early to prevent disease and develop novel therapies to treat the variety of diseases our patients suffer from.

And with that I am very happy to answer any questions. (applause) - Yes, the question is about probiotics in foods and probiotics themselves in over-the-counter products. It is not an area regulated by the FDA. Although they have been trying to focus on companies' claims about what probiotic supplements can actually do. I will also say that not all probiotics are created equal. There are differences in A, the types of microbes found in probiotic supplements, and B, the amounts of microbes, viable living organisms in those products. And that varies enormously between products on the market. I think what's really key are two papers that were published last year that I think were really critical to our understanding of how probiotics can work and they were studies that looked at the gut microbiome of healthy volunteers who consumed a probiotic product.

And what they showed was that whether this species of probiotic was basically allowed to engraft or was able to engraft into the gut was completely dependent on the microbes that were already there. And again, this brings us to the idea that microbes, first come, first served. The microbes that are there already dictate who can enter the party and that was demonstrated very clearly. And that may explain why individuals can respond very differently to the introduction of microbes. Either through probiotics, or through microbes found in the foods we consume. Our microbial encounters and which microbes manage to engraft anywhere in our system appear to be strongly influenced by the pre-existing microbiome that already exists.

So I think we can do better. I think there is good proof of principle and in fact some products have shown efficacy in controlled clinical trials, but I think there is a wide variety of products. They really are not the same. And in fact, there have been some studies that have shown that in some cases some of the products, A, do not contain the species that they are supposed to contain and actually contain a different species that could be harmful to the consumer. So the question is, do you know how the meconium microbiome is explained? Does it come from the mother?

Where does it come from? And if you are going to treat, why treat at birth and why not treat the mother? A few things: Meconium is amniotic fluid that is swallowed. By definition, it is formed in the uterus. In a study that I didn't show you and that we are preparing for publication, it is one in which we really asked when microbial encounters occur in the human fetal intestine. There is much controversy about whether there is a microbiome associated with the placenta. The jury doesn't rule on that. But we are really asking what is happening in the intestine of the fetus.

Because there we know from previous studies, not from our group but from other groups, that the immune system has already begun to evolve and develop. And at 13 weeks of gestation in humans, it has the ability to detect and respond to microbes. So we looked at mid-gestation and found that there was very little microbial signal in human fetal meconium, but we could find microbes there. They're in these little pockets, they're close together. They are embedded in the mucin, so this is not contamination and they are in a subset of the samples we examined. We found an organism whose presence correlated with a specific type of immune cell response.

We were unable to isolate that organism from fetal meconium using media that traditionally select for that type of organism. We had to add pregnancy hormones and a population of immune cells to the selection medium in order to isolate that organism. We have sequenced its genome. It resembles other phylogenetically related organisms, but has unique genomic characteristics that we have not seen in other species, even those that are highly related. Which suggests that it may be highly evolved to be in the fetal intestine in the womb. We think that process probably intensifies later in pregnancy because the communities we detected in postnatal meconium, the first bowel movement after birth, are simple but more complex than the really simple communities we saw in the fetal intestine.

Why not treat the mother? We don't know enough. That's what I would say. Our studies have shown us that, other studies have shown that there is a difference in the microbiome of the three-month-old child related to allergy and asthma, our studies have shown that up to one month of age and in meconium and that is what has led us to The Fetal Gut to ask if there are microbes, but we know virtually nothing about the maternal microbiome during pregnancy. There are shortages of items out there. We know that it changes as the pregnancy progresses. We don't know why that happens.

We don't know what the implications of this are for babies' later health outcomes. And I guess the reason to intervene in early postnatal life is that that's the turning point in microbial development. That is when these communities are at their simplest and when we believe there is greater real estate open to colonization. And rather than ultimately intervene in the pregnancy, but until we know a lot more in that area, it's not something we're comfortable with. We know this and we have many different studies that tell us how high risk of asthma babies are lacking in terms of microbial capacity from the beginning, from the day they are born to the first year of life.

And to me, that's a safer approach rather than playing with what happens in the womb when we have no idea of ​​the implications of what we can do. Yeah, I mean I have to say I would start with the overuse of antimicrobials and their effect on the microbiome. I think there are a number of things that have led us to this point where we have extinguished a number of microbes that we think are probably critical to human function and health. One of them is the plausible use of antimicrobials, but there are many other things. The diet, for example, has changed drastically.

We know that antimicrobial administration causes an acute and sometimes widespread drop in microbial diversity in the gut, but your response to antimicrobial treatment again depends on what microbes you have in your microbiome. Antimicrobials have saved lives. First of all, we need to remember what we have used them for, but in doing so, we may have created a bigger problem with chronic inflammatory diseases. But I will say that I still think they are very useful. In our fecal microbe transplant studies with patients with ulcerative colitis, when we simply performed a single fecal microbe transplant colonoscopy, no one responded to the treatment.

When we pre-treat patients with antimicrobials and then perform colonoscopic delivery followed by a month of treatment, we reach 40% efficacy. So I guess it demonstrates the principle that it clears the way for colonization and that it is useful. And I think a lot of efforts have been made to improve antimicrobial stewardship. I know I went recently with my son who had an earache and they gave us a prescription but told us to wait 24 hours and never filled the prescription. And I think there is much more awareness. We didn't know this field 15 years ago. We didn't know the impact or really think about antimicrobials in terms of antimicrobial resistance.

We don't consider what we are doing to the microbiome with its administration. So I think if anything, it's been beneficial and driven more stewardship of antimicrobials across the healthcare system. There's a great article from Israel a couple of years ago where they studied just that. They studied aspartame, which is an artificial sweetener, and showed that A it affects the microbiome and B that the glucose spikes they found in the patient participants who consumed the artificial sweetener were actually higher than those from a glucose hit. That's right, yes, yes, yes and yes. It affects the microbiome and is really changing the type of metabolism of the system that we think is really what drives the physiology of cells in the human superorganism.

There is very limited information on this, but what has emerged has been quite interesting. They may not be doing what we think they are doing. So, theThe issue is that we have talked about the introduction of fecal matter into children. I haven't, I'm talking about introducing very specific microbes into babies, not feces. What about adults? Great great question. Yes, I mean we believe that the microbiome can be manipulated and redesigned. I think it's a higher bar in established chronic diseases and I think we're starting to understand why. We're starting to look at whether these microbial molecules actually not only change what host cells produce, but they can also rewire the genome of those cells, and we have evidence that that actually happens.

So you're actually undoing several layers of selective pressure on the microbiome from the host side. So I think that's why it takes a month of treatment for children with autism spectrum disorder to see a significant reduction in symptomatology in those children. And I would say that perhaps for those with chronic inflammatory diseases, it could be an even longer-term treatment to control the symptoms of their disease. In some severe cases, it is not clear whether we will ultimately be able to return that system to a healthy system, but we will certainly try. So the question is whether there are studies that have examined the microbiome in adults with obesity.

There is a huge amount of literature on this and I would say there are some of the fundamental studies in the field of the microbiome. Early studies showed that the gut microbiome of obese people is significantly different from the lean microbiome. And even in the simplest terms, just the relative proportions of the key groups of microbes in the gut are sort of Firmicutes and Bacteroides or a ratio of three is to one in lean individuals and is more like 30 to one in obese individuals. . And that's what prompted those studies to ask whether an obese phenotype could be transferred simply by transferring the gut microbiome to those germ-free mice.

If there are approaches to manipulating the gut microbiome to try to undo it, in one of those early studies they did show that an intervention that included a calorie-controlled diet and exercise regimen led to weight loss and a return of the gut microbiome to its normal state. normal state. that 3:1 ratio compared to the obese microbiome. It was a year of intervention. And here's the thing, thinking about what it takes to get to those conditions and the severity of those chronic conditions, I think it's going to be a long-term intervention to really get that system back to something that resembles a healthy microbiome.

The question is whether H. pylori is carcinogenic. I think the really important thing we need to think about here is how these organisms behave and it's completely related to their microbial peers and the conditions of their local ecosystems. And thinking of Helicobacter as a single target to attack, I'm not sure that's the right way to go. Marty Blazer would respond that the loss of Helicobacter Pylori is associated with increased development of allergies and asthma in Western worlds. So I think we need to think a little more critically about what these organisms do, how they function, and really target the function rather than the organism.

Because most of us have Helicalbacter Pylori, but in healthy conditions it is controlled both by the conditions of the stomach and by the organisms that surround that Helicobacter. So I think as we get deeper into understanding the specific mechanisms by which specific microbes drive pathological processes, we become more precise in how we address those pathological processes through microbes. OK, thanks a lot. (applause) (upbeat music)