How Gut Bacteria Influence Mental Health: The Microbiome & PTSD

[00:00:02]Good afternoon and welcome to the Brain and Behavior Research Foundation's Meet the Scientists monthly webinar series.

[00:00:09]I'm Dr. Jeff Borenstein, president and CEO of the foundation and your host for today's webinar. Today, Dr. Christopher Lowry will present the microbiome and mental health. The Brain and Behavior Research Foundation is committed to alleviating the suffering caused by mental illness by awarding grants that will lead to advances and breakthroughs in scientific research. The foundation is the largest private funer of mental health research grants. Since 1987, the foundation has awarded more than $365 million to fund more than 5,000 grants to over 4,000 scientists around the world.

[00:00:53]100% of all donor contributions for research are invested in our grants to scientists who are working to find breakthroughs in disorders such as addiction, ADHD, anxiety, autism, bipolar disorder, borderline personality disorder, depression, OCD, PTSD, and schizophrenia. I'm delighted to introduce Dr. Christopher Lowry. Dr. Lowry is associate professor in the department of integrative physiology and center for neuroscience at the University of Colorado Boulder. Dr. Lowry is also the recipient of a 2007 and 2010 foundation young investigator grant. Today's webinar will begin with his presentation. This will be followed by a question and answer period. To submit your questions, please use the questions tab on the control panel on your screen. Feel free to submit your questions throughout the presentation, but when the presentation is finished, I'll present your questions and we'll address as many as possible in the time allotted. And now I'm pleased to introduce Dr. Christopher Lowry. Chris, the floor is yours.

[00:02:12]Thank you very much. Thank you, Dr. Borenstein for the introduction. Thank you to all the attendees of the web webinar and especially thank you the brain and behavior research foundation for all the the work that you're doing to support research uh to advance mental health treatment of mental health. So um let's get started. Thank you for attending. uh would like to disclose I'm a member of the scientific advisory board of emodulon therapeutics which is a company in London that is interested in use of uh microbiome based products specifically microacteria okay so the outline for today's talk I'll start with the idea of prevention of mental health disorders I'll then go on and introduce you to what what's considered the hygiene hypothesis and the possibility that the hygiene hypothesis may be linked in some way to psychiatric disorders. Third, I'll I'll talk about psychiatric disorders as disorders of a failure of immunereation. So, uh this may be an unfamiliar term immunereation, but think of it as the body's own mechanisms for suppressing inflammation. So inflammation can be a good thing uh when you have an infection but inappropriate inflammation is not a good thing as we understand from cardiovascular disease and we'll also see that inflammation maybe playing a role in in psychiatric disorders as well and then I'll present some data from some research that we've done suggesting that restoring immunereation in individuals that have impaired immunereation can prevent development of a pts D like syndrome in some animal models. I'll then talk about some future research including some clinical trials uh or clinical studies that we're doing in PTSD subjects. So first the idea of prevention of mental health disorders.

[00:04:12]This is a quote from a paper by Tom Insul who was then the director of the National Institute of Mental Health. And over 10 years ago, he and his colleagues raised the idea that in contrast to researchers in cancer and heart disease who've sought cures and preventions, biological psychiatrists in both academia and industry have set their sights on incremental and marketable advances such as drugs with fewer adverse effects.

[00:04:40]Um I'd like to point out that um major depression is common uh and becoming more common. So in this paper in science from 1996, unipolar major depression was ranked number four in terms of disability adjusted life years worldwide and was predict to be number two by the year 2020 ranked only second to eskeemic heart disease. Um back to Dr. Insul and his idea of preventing mental health disorders, they went on to say that psychiatry will need to develop strategies for prevention of each of these disorders. And in that context, they were talking about schizophrenia, mood disorders, and autism. So if we think about the idea of preventing mental health disorders, where should we start? and uh is not a particularly straightforward question.

[00:05:39]Um but we'd argue that we should think about the risk factors for psychiatric disorders. Some of these are well well known in including a genetic predisposition. So the DNA that you inherit from your parents has an an influence on your risk for psychiatric disorders.

[00:05:56]There are also environmental influences and these come in many forms including adverse childhood experiences which you may have heard of as ACE ACEs. Uh but we'd also argue that microbial inputs particularly during early life may be important environmental influences. And here we're talking about the microbiome.

[00:06:19]And so the microbiome is essentially the collection of microorganisms that live on and in the human body. And uh one of the most widely populated parts of the body is our gastrointestinal tract particularly our our large intestine or colon. And so um there can be on the one hand pro-inflammatory microbial inputs.

[00:06:47]So these include bacteria uh or viruses that can cause disease and inflammation.

[00:06:54]But on the other hand, the the side that we don't think of as frequently is a group of microbial inputs that we would consider immunereatory. So if if pro-inflammatory microbial inputs is the the ying, then immunerey microbial inputs are the yang.

[00:07:13]and their um role seems to be suppressing inappropriate levels of inflammation that would be uh disadvantageous to to the host and in some cases to the microbes themselves. Um and so this lack of microbial inputs can lead to a failure of immuno regulation. We'll talk about this in more detail later. And without adequate immunereation we we see increases in inflammation. Uh and we as we'll see in modern urban societies like the United States and European countries there's evidence for uh chronic lowgrade inflammation which is thought to increase our risk for psychiatric disorders. So let's turn our attention to the hygiene hypothesis and psychiatric disorders. Uh this is a paper that we published in 2008 uh nearly 10 years ago uh together with my colleague Graham Rook um who's who's played a prominent role in evaluating the hygiene hypothesis and uh here's a quote from the paper. Some psychiatric disorders in developed countries might be attributable to failure of immunereatory circuits to terminate ongoing inflammatory responses. So, how does that really work in terms of our immune system? Uh, at the top of this photo that you see on your screen now is is what are called the old friends. We'll talk about what types of microorganisms are included among the old friends, but essentially these are organisms that have co-evolved with humans and have adopted this role of suppressing appropriate inflammation. They can do that by binding to immune cells including immature dendritic cells which are labeled DC on the figure. And this drives uh differentiation of these dendritic cells into what are considered regulatory dendritic cells. And regulatory dendritic cells are particularly at inducing the differentiation of T-C cells toward what are called regulatory T- cells or T-reg.

[00:09:18]you can see down in the lower right hand or lower left hand corner of the photo.

[00:09:23]So if I use my pointer, here's the T-reg. And these regulatory tea cells secrete large amount of interlucan 10 and TGF beta. And these are these are molecules that are secreted by T-reg cells that have anti-inflammatory effects. And as such they can suppress inappropriate inflammation in response to self, inappropriate inflammation in response to gut contents and inappropriate inflammation in response to allergens. On the other hand, just having IL 10 and TGF beta around has a general overall anti-inflammatory bystander uh suppression of inflammation. So here's another way of looking at it. Uh our ancestral condition was uh huntergatherer societies, small groups of humans living together eventually uh engaged in agriculture, agriculture and maintaining domestic animals. um living near uh and interacting with these animals and these animals uh and and uh the the lifestyle associated with huntergather and agricultural societies resulted in adequate levels of interlucan 10 and TGFBA these anti-inflammatory cytoines which can terminate inappropriate inflammation and that's associated with the normal ratio of these regulatory toector tea cells. So when we say aector tea cells, these are the ones that are driving inflammation. There can be different types of inflammation which are referred to here as TH1 or TH2 type inflammation that are driven by different types of pro-inflammatory molecules.

[00:11:11]But what we find is that in modern urban societies, we lack the exposure to these types of microorganisms particularly during early life in including the first two years of life and this results in a low ratio of regulatory to affectctor tea cells.

[00:11:29]Consequently, this results uh in increases in inflammation which has been associated with depression, multiple scerosis, type 1 diabetes and Crohn's disease. On the other hand, TH2 type inflammation has been associated with anxiety and allergies. It's worth noting that um there's been a very rapid increase in inflammatory conditions in modern urban societies since about the 1950s to 1960s.

[00:12:02]And so this is a graph from a paper in 2002 in the New England Journal of Medicine showing that the uh incidence of immune disorders has been increasing dramatically since that time and this includes multiple scerosis, Crohn's disease which is type of inflammatory bowel disease, type 1 diabetes and auto uh autoimmune disease and asthma and um early versions of the hygiene hypothes is interpreted these imological changes in urban societies as associated with a shift toward a TH2 type immune response. But it's now clear that essentially all types of inflammation are increasing in modern urban societies including TH1 and TH17 dominated immune disorders.

[00:12:54]Um there has been some limited work comparing biomarkers of inflammation in rural settings like lowland Ecuador compared to the United States. And so this is this these data are from a paper by Tom McDade in 2012.

[00:13:12]and Tom and his team were able to to go to lowland Ecuador and do uh measurements of blood concentrations of C reactive protein. C CRP is a a very good biioarker for lowgrade inflammation.

[00:13:29]It's what the American Heart Association uses uh to determine risk for cardiovascular disease and levels above 3.0 zero are considered levels that confer risk to heart cardiovascular disease. So all these values that we're looking at here are below that threshold by the American Heart Association. But what you can see is that if individuals came into the clinic and they had no infection, their CRP values are very low, less than.5. Um but if they came in with an infection, the CRP concentrations are almost double up to 1.0. zero. But if you compare these values in in people with infections in lowland Ecuador to the average CRP concentrations in the United States in males and females, overall those living in modern urban societies like the US tend to have increased CRP concentrations, which is suggesting uh an exaggerated inflammation under these conditions, albeit a lowgrade one.

[00:14:37]So back to this idea of psychiatric disorders as disorders of failure of immuno regulation. I'd just like to point out that um several neurodedevelopmental conditions and psychiatric disorders have been found to have reduced regulatory tea cells. So again these are the immune cells that have the capacity to suppress inappropriate levels of inflammation. So in this study we'd like to see more studies like this. This was a small study showing that children with uh moderate autism had reduced regulatory tea cells measured in the blood and and children with severe autism had dramatically reduced regulatory tea cells and this goes along with a host of studies showing that there's an increase in uh chronic inflammation in children with autism.

[00:15:28]Likewise, the similar uh observations have been made with depression and post-traumatic stress disorder. In all of these conditions, the the the percentage of regulatory tea cells um circulating in the blood has been found to be reduced. Another argument that would support the idea that there's a lack of immuno regulation in those that are at risk for post-traumatic stress disorder is the finding uh published in 2015 that individuals the diagnosis of PTSD have a higher risk of any autoimmune disorder. You can see that at the top of the graph. and they also have higher risk for some specific autoimmune disorders like thyroiditis, inflammatory bowel disease, and rheumatoid arthritis.

[00:16:17]This is consistent with the idea that there's simply a lack of the ability of the body's own immune system to suppress inappropriate inflammation. In this case, inflammation that's directed against self antigens leading to autoimmune disease. Another line of evidence is this study that was published in 20 2014. Um this was a group of uh nearly uh well 2,600 war deployed Marines and in this case they measured CRP again this is a biioarker of inflammation uh at boot camp and again post deployment and what they found is that indiv so remember this is the cutoff for the American heart dise heart heart association for cardiovascular disease 3 milligram per liter what they found is that individuals that had higher CRP concentrations at boot camp had higher risk for development of PTSD symptoms after deployment.

[00:17:20]So if in fact inflammation and this lack of immunereation is a risk factor for development of PTSD, would it be possible to to provide an intervention, restore the immuno regulation, suppress inappropriate levels of inflammation and pre prevent a development of a PTSD like syndrome? To address that question, uh we used animal models. But before going into that, I want to talk about what these old friends are. So there's three general categories of old friends that can suppress inappropriate inflammation.

[00:17:54]The first is what we would consider the the human microbiome. So these are organisms that part of the co-ol human microbiome. They include um microorganisms like bacteritis fragilus lactobacillus rutery that you can buy as probiotics. Uh acamia mucinophila is a a highly specialized bacterium that eats mucins in the mucous layer lining the gut intestine and um has been associated with um driving immunereation and suppressing inflammation. Another example uh perhaps somewhat surprisingly is what are called the old infections. So these were are infections that were present during the hunter gather period of human evolution.

[00:18:40]uh usually harmless transmitted by the fecal oral root very early in life that have been depleted since urbanization. So there's um evidence that uh parasites played this role hepatitis A virus toxopplasma gandi some strains of te gandi salmonella and h pylori we'll come back to h pylori later the third category which I'll I'll focus on today is the harmless environment environmental organism in mud untreated water and fermenting vegetable matter these are some sometimes called pseudo commensiles they include include lactobacilli and environmental sapites. Saprophy is bacterium that lives off of decaying organic matter and that includes microbacterium backy which I'll be talking about uh and showing you some data later in the talk. In general, our exposure to all of these or classes of organisms has been reduced in modern urban societies. So let's talk first about the first category of old friends, the human microbiome. This slide is just to make the point that our microbiome in modern urban societies is thought to be quite different from what we would consider to be a more ancestral microbiome that you can find in hunter gather societies like the anami uh amarindians guibbo uh population both of these in in uh upper Amazon basin in Venezuela and Colombia and also the Malawi uh populations and so there's a consistent alignment of these huntergather populations in terms of the community structure of the microbiome. So these are fecal samples um and simply looking at all uh let's say 500 or 800 different uh species that are found in the microbiome. Looking at these communities and we'll we'll see a few of these plots later. These are PCOA plots and what this what this is showing is that uh if samples are close to each other like these two their their microbial communities are very similar. If samples are far apart in Pacebased these communities are very different. So what this is telling you is that the the US uh microbiomes from US individuals cluster together and they're very different in terms of their community structure to huntergather societies. Um this is a a separate type of metric of the human microbiome. This is bacterial diversity phoggenetic diversity or PD. And in general we think higher diversity. So here on the y ais scale is uh a healthier microbiome and uh today the the um population with the highest alpha diversity of gut microbiome is the yanami Indian population in the upper Amazon basin. They have a diet that consists of wild, bananas, seasonal fruits, plantains, palm hearts, casava, birds, small mammals, fish, small fish, crabs, and frogs. So what um we know is that stress decreases the diversity of the microbiome. Um and that provides opportunities for opportunistic pathogens uh to get a foothold and to cause infection and disease. So that's been established experimentally by Michael Bailey and his colleagues. Um and and so simply looking at alpha diversity, it it looks as if those in the United States population have reduced diversity and this may be a less stable microbiome and perhaps not not as resilient as as you might find with populations with higher diversity. So, I've I've uh included my own microbiome report from the American Gut Project.

[00:22:50]You can see the URL for the American Gut Project down here in the in the lower right. You can you can also have your uh gut microbiome analyzed by the American Gut team, Rob Knight and his colleagues.

[00:23:04]Uh simply they'll send you a kit. You take a swab uh of uh stool and then put that back in a sterile tube and send it back to them. They analyze it and they send you back a report like this on your own gut microbiome. So you can see here's my sample on the left and as is typical for Americans um my sample has a relatively equal uh abundance of firm what are called firmicutes and bacteroids. And so these are two fila of bacteria the most abundant filyla in uh American populations and in general the this is in the order of abundance. So firmicutes tend to be the have the highest abundance. This includes the lactobacilli that you find in probiotics like yogurt um kombucha and other other foods. Uh and then bacteroids is next.

[00:24:00]Proteacteria third. These are of interest because they are gram negative and contain lipopolysaccharide in the cell wall which can be potently pro-inflammatory. And then uh fourth is actinoacteria. This is the film to which the environmental sacrifice uh belong including mcoacteria which be talking about later. This is a rare abundance film but we think it may be particularly important as I'll show you later in the talk. So here's average relative abundance of different filyla in in uh the American gut project sample. Here's um comparison with someone with a similar diet uh males only in my case similar BMI similar age and mopollen. So, Michael Pollen um has written uh and spoken a lot about uh diet and uh I think he's quoted as saying uh mostly plants not too much kind of summarizes his philosophy.

[00:25:06]Um so in my case and micropollen's case um by far the the the microbiome's dominated by firmicutes and bacttoides and not not so many proteobacteria. So you also get a of how you um align in in PCOA space with other samples in the project. And so in brown are the fecal samples in blue dark blue are the oral samples from the mouth. And um in dark green are are samples from the skin. You can see that this is a a fundamental property of the microbiome that the fecal microbiome is different from the skin microbiome and and that's different from the oral microbiome.

[00:25:52]This plot shows how uh in my case I compare with other individuals in my in in my country the United States uh and those with the western diet emmerindians which is hard to see on the screen but they're down here and also Malawians which are spread spread out here in this PCI plot and then finally there's some attention that's been given to the the firmutes to bacterroid ratio and here it's showing vermicutes abundance and my sample is somewhere here in the middle uh with about 50/50 which is pretty typical for modern urban society. So uh the American Gut Project now has over 10,000 samples. Uh they've summarized some of their findings here which again you can find on their website.

[00:26:40]Um they have a large number of samples as I said over 10,000. The your microbiome changes as you grow. As you get older, your microbiome becomes more diverse. Um, we know that antibiotic usage affects their microbiomes, reduces diversity, uh, leading to a less healthy gut environment. Uh, and this is an interesting finding that the more different types of plants a person eats, the higher their gut microbiome diversity. I think on the survey that they're using the highest responses I eat uh greater than 30 different plants. Um and this is interesting because we know that plants have their own microbiomes. Uh these the the bacteria that live inside plants are called the endopites. Uh, and an interesting piece of trivia is that a three to four leaf spinach plant has over 800 different species of bacteria inside the plant that you can't wash off. And so, um, just like humans, plants have microbiomes and when you eat fresh plants, you're eating the microbiomes of those plants. So, one, uh, approach to increasing diversity of microbiome may be to eat more plants. Um and also alcohol consumption also affects microbiome diversity. Uh those who had a at least one drink per week had a more diverse microbiome than those who abstained from alcohol. Okay. So that that kind of summarizes the the um the commensal organism. What about these old infections? This is the second category of old friends. And I want to discuss a case study which is helicoacttor pylori.

[00:28:24]It's the most common bacterial infection worldwide. It co-evolved with humans at least since humans migrated out of East Africa 60,000 years ago. And um originally Hori came to fame because it was discovered that it's a causal agent in uh datadinal ulcers um which were very common uh several decades ago and in fact uh the person who who discovered that won the Nobel Prize. It's since been discovered that H. pylori particularly during development has potent immunore regulatory effects. So in a sense you can consider h pylori uh an old infection. I'll show you how um that works in a in a moment. So here's the iminology. I don't expect anyone to um understand all of these uh abbreviations here, but just like uh I showed in an earlier cartoon, the bacteria is binding uh and interacting with what are called regulatory dendritic cells. This results in the release of anti-inflammatory cytoines like interlucan 10 and GGFBA. and having interlucan 10 and TGFBA around tends to bias the differentiation of TE-C cells toward these regulatory TE- cells that produce anti-inflammatory cytoines and by biasing toward the production of regulatory TE- cells. This limits the production of TE- cells that drive inflammation like the TH1 cells and TH7 cells that can cause immune disorders.

[00:30:06]So I H pylori is able to bias um the the differentiation of TE- cells toward regulatory TE-C cells and dendritic cells that have have been exposed to H pylori tend to be poor inducers of these pro-inflammatory immune cells. So overall we would consider H pylori uh if present during development have imunore regulatory and anti-inflammatory effects. Here you can see uh what's happened um in terms of H pylori prevalence. Um this is showing uh prevalence of H. pylori infection in various uh underdeveloped countries um compared to the United States and as you can see especially during childhood um there's a much higher prevalence of H pylori infection in uh these non um US countries and of particular concern the the uh exposure among childbearing women is low suggesting that uh even women during childbearing years may have low um immunore regulatory inputs. So the US population is very aligns very well with uh other urbanized countries including those in in Europe and Japan. So uh having introduced first the the the commensal microbiome then the old infections now I'd like to turn to the third category of old friends which are these environmental pseudo commensiles. They're called pseudo commensiles or false commensiles because throughout human evolution they would have been present in high numbers high quantities in the drinking water uh for example but they don't colonize tend to colonize at least the gut mucosa. Recent studies have found that microacterium uh in particular are highly abundant in the airways. And so uh the the the lack of detection of microacterium in the microbiome may simply be a a result of most the fact that most studies study the gut microbiome as opposed to the the micro microbiome of the airways. Um so one of these microacteria is microacterium vaky. Vaky is uh based on the Latin word for cow and based on the fact that this this uh species was originally identified in cow dung. Uh but it's an envi an immuno regulatory environmental sacrifice. So normally it lives off decaying uh organic matter.

[00:32:54]Microacteria tend to be high in in soils that have high organic uh content. So pedy soils, black swamps, that kind of thing. Um, and the history of m this particular strain of microbacterium goes all the way back to the 1970s when John Stanford and his colleagues noticed that um in clinical trials for uh vaccination against leprosy the efficacy of the vaccine depended on the geography. So in some geographical areas these vaccines were highly successful. In other geographic areas they were not very successful at all. And so they went to an area around Lake Kyoga and Uganda uh which was an area where these vaccines were highly successful with the idea of trying to find some environmental factor that could explain why these vaccinations were so highly successful. When they got to the lake, they found that the shores of the lake were lined with this orange slime. And the orange slime turned out to be mcoacterium vaky, which is shown here in a scanning electron microscope image.

[00:34:06]And so, uh, people living around the lake would be exposed to high concentrations of microacteria, using that for drinking water, for cooking. Um and uh as we'll see later on, these bacteria don't have to be living to have an effect on the immune system.

[00:34:27]Um so we then wanted to ask the question if we immunize in our animal models if we im immunize animals with a heat killed preparation of this mcoacterium vaci which was subsequently found to have uh these potent immunore regulatory effects and anti-inflammatory effects can we then prevent development of a PTSD like syndrome so uh after discovering ing that microacterium uh was in fact important in altering the efficacy of these vaccinations against leprosy. Uh this was because uh microacterium backis closely related to microacterium lepre uh and mbback appears to be acting as an adimant in addition to that it also has these immuno regulatory properties. So it increases the differentiation of regulatory tea cells and production of anti-inflammatory cytoines. So we wanted to test this in a model uh of chronic stress to see if we could prevent the development of outcomes of chronic stress. And for this we chose to work with uh Dr. Stefan Reber who at the time was at University of Regensburg in Germany and now is at the University of M in Germany. Um this is a paper describing um the potential use of of the experimental model as a model of post-traumatic stress disorder. I won't talk about why we think it's a good model for post PTSD, but uh I've shown you the paper here if uh you're interested. So the model consists of uh a a large dominant mouse co-housed with four subordinate mice. So in nature, mice uh are social animals and they spontaneously establish these dominant subordinate hierarchies much like primates. And uh in this particular scenario, the dominant mouse will quickly uh uh exert dominance over these smaller mice. Smaller mice show these stereotypical and species stereotypical uh behavioral responses including what's called upright defensive posture where they stand up uh and freeze and that's a signal to the dominant mouse that they are in fact subordinate um and to leave them alone um and that they're not going to be a threat to the dominant or the dominant status. So the the chronic subordinate colony mice, the CSC mice, uh are stressed in this protocol and um I'll show you in a moment what um those stress outcomes are. But this is the general design for our experiments where we immunize animals once a week for 3 weeks. And then uh we expose them to this chronic subordinate colony housing model which is 19 days. And then we do behavioral testing for anxiety and collect tissues for analysis of tissues. So uh the controls for these studies are single house controls. We use single house controls because uh mice spontaneously form dominance hierarchies even if they're all the same size. Okay. So what do we see in this model? We see evidence of uh changes in the adrenal gland adrenal hypertrophy.

[00:37:57]So the adrenal gland grows. This is a common response to stress in mammals. Um cells lose their responsiveness to gluccocorticoids.

[00:38:10]um invo and in vitro and then the sensitivity of im immune cells to to the stress hormones gluccocorticoids is also decreased. There's also increase in anxiety as measured by a number of tests and evidence of some some changes in uh related to sematic disorders including histoologgical damage to the to the colon. This is a spontaneous colitis and so there's inflammation in the colon. Uh there's increased risk for inflammation related colon cancer and there's in increased risk for of severity of chemically induced colitis which is a model of inflammatory bowel disease.

[00:38:54]So when we conducted these studies, we injected a whole heat killed microacterium beaky three times as I I mentioned and then we looked at their behavioral response during the first very first hour of exposure to the dominant male. And what we noticed right away is that mice that had been immunized showed a dramatically different behavioral response to this stressor. and specifically they showed decreased um submissive upright postures. So we we interpreted this as a a shift in their behavioral strategy toward a more proactive response to the stressor.

[00:39:36]Interestingly uh a passive or subordinate response during trauma in humans is also associated with an increased risk for PTSD uh later on. And so the mice were still subordinate as indicated uh here by a negative score, but they were far less subordinate than the control animals.

[00:39:59]And they were far more likely to show proactive behavioral responses, either chasing or attacking the dominant uh male in the cage. So we we wanted to look at uh the gut microbiome in in the stressed animals and the animals that had been immunized. And what you can see is this alpha diversity in its simplest form this can be viewed as a number of different species in the gut microbiome. And you can see that in all groups the the diversity goes down after stress exposure. And this is a very consistent finding with stress exposure uh in animal models. um by Michael Bailey and his colleagues and and others. Uh and there's some evidence for uh a protection from the stress induced decline in diversity of the gut microbiome in individuals that were immunized. If we look at the beta diversity, uh time is on a fixed axis. So these are the three immunizations before the stress starts and uh this is day one before the stress starts. This is day eight and day 15. And after stress begins, which is shown here with this gray shading, you can see this kind of expanding universe of of beta diversity of gut microbiomes. In other words, what's what this is saying is that um after stress exposure, all the microbiomes of the individual mice getting different from each other in almost stochcastic or unpredictable ways. And so you see this expansion of beta diversity among all the animals that were stressed.

[00:41:45]um and we did find an effective immunization decreasing the stress induced increase in beta diversity. So we would interpret that as a stabilization of the gut microbiome. So it doesn't prevent changes in the gut microbiome, but it it had a small stabilizing effect. Here's another way of looking at what stress uh what what impact stress had on the microbiome. And you can see that stress on day 8 and 15 among the CSC mice induced a massive increase in proteacteria. Specifically, this was driven by bacteria that belong to the genus helicoacttor ACA or family helicopactor ACA genus helicoacttor. And you can see that the immunization in red did not prevent the stress induced increase in helicopactor.

[00:42:42]And notice that this is several orders of magnitude increase in helicoacttor abundance. Uh we think this is very important. We know from other groups that gluccocorticoids the stress hormones like cortisol in humans or corticostone in animals can increase the proliferation of helicoacttor.

[00:43:02]um and this is mediated by the gluccocorticoid receptor. So it may well be that simply being exposed to stress induces the proliferation of helicoacttor. Um in our case the immunization did not prevent this but what's interesting is that it prevented um the colitis and so you can see here is a stress induced colitis an increase in inflammation in the colon and this was attenuated or prevented in the individuals that had been immunized with microacterium. This is a PCA plot showing that uh this is before stress the open symbols. This is after stress the closed symbols. And these vectors are showing which which um species are driving this stress induced shift in the microbiome composition and you can see it's dominated by helicobactctor species. In contrast, uh this genus mucus spirillum goes down and mucus has been suggested uh in other studies to be a biioarker of gut in uh infection. And so in the presence of gut infection um the the mucus layer is disrupted and consequently mucus which feed on the mucus uh have don't have a niche in the gut microbiome and they disappear and they only come back after the gut infection resolves. So here uh is a study looking at this chemically induced colitis. You can see that stress increases chemically induced colitis in this model of inflammatory bowel disease and this is prevented by immunization with microacterium becky. So importantly stress induces um from meantic lymph node cells. So these are immune cells that have been isolated from lymph nodes that are draining your gastrointestinal tract. Um in this case these immune cells were um releasing large amounts of interferon gamma and pro-inflammatory cytoine. Um and this inc stress induced increase was prevented by the immunization. Again a massive increase in interlucan 6. This was prevented another pro-inflammatory cytoine. This was prevented by the immunization. And perhaps just as important in instead of producing interlucan 6, animals that were immunized were producing large amounts of interlucan 10. Notice the scale here. Uh these are massive increases in interalucan 10 and anti-inflammatory cytoine. And so the immunization although it's not preventing changes in the microbiome, it's preventing pro-inflammatory responses that we think are driven by the microbiome. So it's allowing the host to tolerate what we might consider as a microbiome with high uh potential for inflammation. So uh just a few more slides. I know we're coming to the end here. We've done a a small exploratory study with Sean Hemmings and her colleague colleagues at Stellenbos University in in South Africa. In this study, we're looking at PTSD subjects and trauma exposed controls in South Africa. And so, everyone was exposed to trauma, but as is typical, only a subset of individuals exposed to trauma go on to develop PTSD. And what we found was that individuals that had uh high relative abundance of bacteria and baruko microbia which include included mainly acium mucinophila had a very low risk of PTSD. So on in in contrast having low abundance of bactinoacteria like which includes microacterium backy for example um was associated with a higher risk of going on to develop PTSD. This may give some evidence of of mechanisms involved. This was a study done by Carl and colleagues just recently published showing that high relative abundance of active bacteria is associated with low gut permeability in response to a a stressor in this case a 51 kilometer ski march. Um what about our uh future directions? Where are we going in the future? Um I'd like to point out the showerhead microbiome project. We're um doing this with Noah Fear here at CU Boulder, Rob Dunn at NC State, Nate Sanders, uh University of Copenhagen, and Jen Honda and Ed Chan at National Jewish Health. Um and so we're we're studying the shower head because we know that showerheads and municipal water supplies are a major source of microacteria. So if you look in soils, microacteria abundance is around 1%. But if you look in showers and municipal water supplies, it's about 15%. And and can be as high as 98%. And so we're looking at hundreds of samples of showerheads across the United States uh and the rest of the world in order to find out what types of microacteria are found in municipal water supplies. um and and uh are are they correlated with uh any any uh elements of the water chemistry or other factors that we can identify? And then we're also uh in a project funded by the Alfred P. Sloan Foundation looking at aerosolized mica.

[00:48:48]So in these studies the bacteria are simply being aerosolized and we're allowing the mice to breathe macteria.

[00:48:56]Uh so they're being aerosolized from a water source and this would be very similar what you might experience in your own shower where the shower head is aerosolizing microacteria and then you're breathing it in. Um and in fact Noah and his colleagues have found that microacteria abundance is higher in homes than it is outside the homes and that it's thought that these microacteria are in fact coming from showerheads and other water supplies. So in conclusion, exposure to what we would call immunereggulatory old friends has declined dramatically in developed countries in the last 50 years.

[00:49:31]Psychiatric disorders are associated with decreased uh regulatory tea cells, decreased immunereation and increased inflammation. We found that immunization with microacterium prevents stress induced inflammation and anxiety and fear responses. And this suggests that microbiome based interventions, sorry about the typo, to increase anti-inflammatory immunereatory signaling might be considered for prevention and treatment of psychiatric disorders. So with that, I'll end and I'll take any questions that um attendees of the webinar might have.

[00:50:04]Thank you very much for your attention.

[00:50:06]It's been a pleasure. Chris, thank you very much um for the work that you do and are doing and for an excellent presentation on a complicated topic.

[00:50:17]Um, one of the the questions that a few people have asked is if there's somebody who does have a psychiatric condition, um, is there something that they may want to do um, to affect inflammation, you know, visav diet?

[00:50:35]Yeah, that's an excellent question and um I I would say I would say yes to the extent that inflammation may be a risk factor, there are ways to to uh including lifestyle choices to decrease inflammation. So, there's a number of anti-inflammatory diet protocols that have been published. Um you may want to find one of those. Um, you know, good a good example is if you eat a hamburger, you have a very rapid inflammatory response. But if you include a slice of avocado on the hamburger, uh, it suppresses that inflammation. So these dietary choices that we make are very important for inflammation. Um, also exercise is important, sleep is important. Um and so these are life lifestyle factors not to mention u you know aspirin or other um anti-inflammatory drugs that that could have an impact as well the with regards to potential steps for prevention. What types of things do you see coming in in the in the short term?

[00:51:44]What what where do you see the research bringing us in terms of interventions for prevention?

[00:51:50]So clearly lifestyle factors um should be on everyone's mind because that's something that is accessible to most people uh if they're informed and um another alternative may be probiotics.

[00:52:07]We're finding that many probiotics in other words live bacteria that confer a a health benefit for the host that you find in yogurt and kombucha. um many of these probiotics as long as they have immunereggulatory and anti-inflammatory effects may be beneficial. That hasn't been fully demonstrated yet by any means. I would say we're conducting a clinical trial with Lisa Brener at the VA in Denver uh looking at an amunore regulatory probiotic in uh PTSD subjects with mild traumatic brain injury. So those types of trials are ongoing and hopefully within a few years we'll have more information about potential impacts on stress resilience and other outcomes. And above and beyond what you described, is there any work that's looked at different general diets, you know, a vegan diet and other diets that people have that may sort of provide some information about this?

[00:53:11]Uh that's an excellent question. Um I'm not aware of this is not to say that hasn't been done. I'm simply not aware of uh studies looking at comparing the diverse types of diets that you've mentioned. Uh I am aware of some studies using some some diet uh dietary interventions like omega-3 fatty acids which have shown some promise and so diets high in these types of fatty acids may in fact be beneficial.

[00:53:39]uh but there's a lot more work to be done and that that was in the context of depression. So this is in the context of psychiatric disorders.

[00:53:46]Right. Right. And you speak about the issue of stress which we've you know in our field we look at in in general but you're really coming at stress from a different direction in terms of the the the role uh of stress and inflammation.

[00:54:04]Um, and I'm just curious where you see future directions of that in future research. Yeah. Yeah. I think it it we're on the verge of a paradigm shift, I think, because when we think in in classic terms of of stress, we think of activation of the hypothalamic pituitary adrenal axis and gluccocorticoid hormones. We think in terms of activation of the sympathetic nervous system, but it's been less well recognized that there's also an inflam inflammatory response to stress. And the potential relevance to psychiatric disorders, I think, is illustrated most clearly by uh a study by Tad Pace and his colleagues where they they looked at a a social stressor a true social stress test uh and then evaluated interlucan six response in depressed patients and healthy controls. So remember IL6 isn't is one of these pro-inflammatory cytoines and what they found was that there's a massive increase in interucan 6 in depressed patients. It's well well above that you would find in a healthy control individual. And so most studies that have done metaanalyses of inflammation in depression and schizophrenia, PTSD and other psychiatric disorders have found these low this low evidence for lowgrade chronic inflammation. But I I would suggest that's really just the tip of the iceberg. And what we're missing is that the fact that the whole immune system is lacking immunereation and consequently when a stressor comes along there's a hyperactivation of the immune system and a hyperinflammatory response and we don't often test that in our in our clinical studies but I suspect that that's the case.

[00:55:47]the well again I want to thank you Chris for your ongoing work in what you know may really help change our approach to treatment and prevention in a significant way. Uh thank you for taking the time to do uh such an outstanding presentation for us um and uh uh look forward to hearing um more as you continue the work down the road. So, thank you very very much. Um, I also want to thank our audience for joining us today. All of the research that we fund is made possible through private donations. And if you'd like to make a contribution, please visit our website at bbrfoundation.org or call us at 1 8008298289.

[00:56:38]The webinar has been recorded and if you've missed any portion of the presentation or would like to share it with a family member or friend, please visit the events page on our website.

[00:56:49]Um, finally, I hope people will join us again next month when Dr. Dolores Malispina will present a webinar entitled Searching for the Seeds of Psychosis. This will take place on Tuesday, August 8th at 2 p.m. Eastern time. Once again, thank you for joining us and enjoy the rest of the day. Take care.