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Webinar #5-- The Microbiome and Mood

Guy Daniels

This educational webinar relates to just about everyone-- learn how your gut microbiome plays a role in your daily mood, levels of stress, and feelings of anxiety and depression in our latest must-listen webinar by Onegevity's Guy Daniels. Subscribe to our YouTube channel to catch all of the education from this series!

Slide 1 – Welcome to another webinar in our comprehensive series on the microbiome. My name is Guy Daniels, and I’m the head of medical education for Onegevity Health. Today we’ll be discussing the microbiome as it relates to mood, which will encompass stress, anxiety, depression, and PTSD.

Slide 2 – Our first slide may look familiar to those of you who watched the webinar entitled “Let’s Start at the beginning.” In this large study, it was shown that reported stress, via a subjective questionnaire and salivary cortisol, an objective, measurable form of stress, during late pregnancy, were associated with dramatic shifts in the infant's microbiome, which continued through follow-up out to 16 weeks. What were these shifts? As you can see from the figure to the right, “A” represents the low-stress microbiome, while “B” represents the high stress. The obvious jumps out at us that there is a significant increase in yellow and red in a high stress, which represents the class Bacilli and the phylum Proteobacteria. Although Lactobacilli are in the class Bacilli, we’ve found they aren’t always beneficial.

Additionally, there are other potential trouble-makers in this class, to include names with which you’re beginning to become familiar, like Streptococcus, Staphylococcus, and Enterococcus. And of course, we all now know that Proteobacteria is full-on opportunistic pathogens, and is a phylum we want less of in our reports. This dysbiosis caused by maternal stress during pregnancy passed along a less desirable start in life for these infants, which resulted in more allergies and gastrointestinal symptoms.

Slide 3 – To continue our dive into stress, we have this interesting study from Japan, looking at how the stress of a cadaver course, impacts the microbiome of medical students. The study design is shown on the left, which is a double-blinded, placebo-controlled, parallel, and crossover trial. The researchers simply administered a probiotic called Lactobacillus gasseri, or a placebo, and measured the fecal microbiome. As you can see from the right side, there were significant differences in the trouble-making family Enterobacteriaceae, and the potentially troublesome genus Veillonella, between the students who took a placebo, vs the probiotic. Just by administering one beneficial species, the significant rise in stress-induced dysbiosis was negated. And the tangible benefit was better to sleep, and less abdominal pain, than in the placebo group.

Slide 4 – I’m going to present to you one more slide on stress, and then we’ll dive into what’s going on a bit behind the scenes. In this interesting two-part study, the researchers measured the effects of NSAID use (which is known to cause GI permeability), the anticipation of electric shock, and public speech, both stressors on the HPA axis, on GI permeability, via a very standard test. In part two, they took a deeper dive and administered CRH (a stress hormone) and a mast cell stabilizer, to determine the mechanism of action. They found that NSAID use and public speech increased intestinal permeability and that the salivary cortisol, was only increased in public speech, and the permeability was prevented, by using the mast cell stabilizer. So this tells us two things. People really hate public speaking, and the detrimental changes that were initiated by stress were manifested via mast cells. So let’s take a look at some of what’s going on here.

Slide 5 – I’m going to go out on a limb and say that most of us won’t be speaking publicly often, and if you’re routinely exposed to electric shock, you should reevaluate life choices. However, most of us experience daily stressors in one form or another. Some are stressful, but not terribly acute, and others are more severe in nature. Frankly, we didn’t evolve with the capacity to deal with the crazy lifestyles we lead these days, at least they are crazy from a biochemical viewpoint. And whether someone knows the science behind stress and illness or not, we all know that there is a connection between the two. So on this slide, we revisit a topic from our webinar on IBD, that of iron. As I stated before, this relationship between iron, bacterial growth, and virulence, has been known for many years, with the limited availability of free iron in the host environment, presenting a major obstacle to the growth of most microbial pathogens. The body sequesters iron in lactoferrin and transferrin, in an attempt to keep it away from opportunistic pathogens. Now you have to understand that these bacteria are in all of us. There are many opportunistic pathogens out there, and you will have a number of them in your gut. The question is, what is the overall balance of the good bacteria vs. the not-so-good. In times of stress, and here we’re talking more acute stress, the adrenals, and the enteric nervous system release what are called catecholamines, which are your dopamine, L-dopa, norepinephrine, and epinephrine (think adrenaline), into your bloodstream, which enables several opportunistic pathogens. They do this in a couple of ways. One of which is related to virulence. Catecholamines can increase the toxicity of some species. For example, norepinephrine increases an adhesion protein for E.coli. Remember, we don’t want these guys making their way to the cells that line our gut, the enterocytes, adhering to them, as is the case here, and invading them. The other manner in which the catecholamines act to serve pathogens is in freeing up iron. A portion of the catecholamines, and their metabolites, complex with the ferric iron sequestered by transferrin and lactoferrin, thereby reducing the iron-binding affinity of the proteins, and rendering them susceptible to bacterial ‘theft’ by ferric ion-specific chelating agents, produced by the pathogens. For Gram-negative bacteria, such as E. coli or Salmonella, the synthesis of these agents, and uptake systems are integral elements in the mechanism by which stress hormones induce growth. And that’s what we see here on table 1 to the right. We see many plus signs in the growth column. And in the virulence column, which we touched on previously, we see more. The species listed here are largely a who’s who of opportunistic pathogens, all of which have been shown to be responsive to stress hormones, as listed in the references. 

Slide 6 – If we look at just one of these opportunistic pathogens, Campylobacter jejuni, which by the way is in the concerning phylum Proteobacteria, we see in this paper, that norepinephrine, does indeed stimulate bacterial iron uptake and growth, in iron-restricted conditions, imposed by the high-affinity iron-binding glycoproteins transferrin and lactoferrin. In the case of pathogenic bacteria, effective iron acquisition in the face of the defensive iron-sequestering response of the infected host may be crucial to the outcome of an infection. These bacteria use a variety of mechanisms to acquire iron, including ferric reductase activity, interactions with host iron-binding proteins, and the use of the previously mentioned low-molecular-weight secreted molecules, with high affinity for ferric iron. Many species possess the genetic capacity both to synthesize and to take up molecules, and some species express uptake systems for those secreted by other microorganisms. By the way, not only is Campylobacter jejuni a possible food pathogen, but about 95% of us have an identified low abundance in our guts.

Slide 7 – There are many other ways stress and dysbiosis can affect one’s mental health. As I’m sure you know, the gut talks to the brain and the brain talks to the gut. The state of dysbiosis can impact the robust immune system which lines the GI tract. When stimulated, the immune system produces an array of compounds which are largely inflammatory. These compounds not only have implications within the gut but also systemically. For example, A meta-analysis of cytokines in major depression, looked at twenty-four studies, involving measurements of these immune-generated inflammatory compounds in patients with major depression. There were significantly higher concentrations of two of the key cytokines, TNF- alpha and IL-6, in depressed patients. So this is a reflection of chronic low-grade inflammation affecting for the purposes of this webinar, brain function.

Slide 8 – This chronic low-grade inflammation, is characteristic for many conditions, including diabetes, cardiovascular disorders, autoimmune diseases, chronic fatigue syndrome, depression, and neurodegenerative pathologies. If we take a deeper dive into this immune-driven inflammation, we find a classic player both within, and outside the gut, via what are called PAMPs, or pathogen-associated molecular patterns. These are molecules recognized by our immune system, as something to be alarmed about. The most studied one of these is called LPS, aka lipopolysaccharide, or endotoxin. This comes from the cell wall of opportunistic pathogens. So let’s follow one possible chain of events. You have dysbiosis from a variety of lifestyle factors, which means you have more trouble-makers in your gut, and fewer good actors, to keep them in check, which in turn increases inflammation in the gut via the immune system. One of the many molecules produced in inflammation is the much-researched TNF alpha. TNF-a then in turn via enzymatic activity, reorganizes the tight junctions in your gut to make them more permeable, which allows contents from within the lumen of the gut, to pass through that critical layer, and enter into the bloodstream. One of those molecules in LPS, and as you can see from table 1, serum LPS plays an enormous role in many diseases. It does this, evidently primarily, by being recognized by what are called toll-like receptors, or TLRs for short. In this case, we’re focused on TLR4, which plays a special role in recognizing LPS. The subsequent immune activation due to the alarm going off, in recognition of an invader, results in a large suite of pro-inflammatory molecules being released to combat the said invader. This is all well and good if, for example, you ate some contaminated food, and once cleared, this process is self-limiting. However, this is very bad news in the chronic state, as we’ve seen in the IBD webinar, chronic inflammation can result in terrible outcomes. TLRs are also expressed on different cells types outside the gut, including immune cells, adipocytes, and endothelial cells.

Slide 9 – And as we can see on this slide, LPS can activate Toll-like receptors, which are also present on enteric neurons (that’s nerves in the gut), sensory afferent neurons in the spine (afferent means their sending signals to the brain), and various cells directly in the brain, modulating their activity, and affecting the function of both the enteric nervous system, and the central nervous system. We’ll learn more about the role of LPS in our next webinar on metabolic syndrome, which research has abundantly shown is related to dysbiosis. Consider, that we’re talking about just one facet of dysbiosis here, albeit a well-proven one. There are many more molecules, immune factors, receptors, proteins, and more, many of which we’ll gradually cover in our series. 

Slide 10 – Returning to the role of the gut in anxiety and depression, here we have a diagram which incorporates several factors within this webinar. At the bottom in light brown, we have the lumen, or interior of the gut, with its array of bacteria, hormones, and the ever-important SCFAs. These, in turn, interact with the many cells which line the gut, which among other things produce many messengers, such as neurotransmitters and hormones, and components of the immune system. In the middle, we see a direct line of communication to the brain, the vagus nerve, which is yet another way the gut talks to the brain. With its connection to the brain on one end, and the enteric nervous system on the other, you can appreciate its role; when you consider that the enteric nervous system interacts with the microbiome, thus the whole complex is referred to as the gut-microbiome-enteric nervous system-vagus-brain pathway. Not only can the microbiome generate transmission through this pathway, its byproducts, have been shown to actually pass through the vagus nerve, and make their way to the brain, affecting brain function, stress responses, and sleep structure. For example, ammonia, from excessive protein fermentation, is one such neurotoxic metabolite, which can have detrimental effects. Another is D-lactic acid, which is one of the forms of lactate, formed by several bacteria, to include lactobacillus. So, if you see in our report, a genetic propensity to be an over-producer of lactate, and we know lactate accumulation is not a good thing, then we can make recommendations to drive the necessary changes in your gut, to decrease lactate producers and/or increase lactate consumers.

Slide 11 – So what might some of these recommendations look like? Here, we have a systematic review of the literature from 2019. These researchers eventually narrowed their focus down to 21 studies, looking at modifying the microbiome in anxiety. What they found, was that just over half of the studies showed that modifying the microbiome via one of a few mechanisms, was able to improve anxiety symptoms, with food and prebiotics faring significantly better than probiotics. So here in our webinar on mood, we illustrate a point I’ve brought up before, that of diet, and especially prebiotics being the engine that drives the change. Again, this is not to say that there is no beneficial data for probiotics, which we saw in the aforementioned Lactobacillus gasseri study. I’m simply saying that with probiotics, we are dealing with some unknowns with multiple species, lactobacillus being the best example, we’re dealing with administering only one or a few species instead of changing dozens or more, and we’re dealing with lower doses, which can be driven harder by multi-gram doses of prebiotics, as we see here in this study.

Slide 12 – This is a case for using prebiotics in anxiety. In this 12-week parallel crossover clinical trial, 44 patients were randomized to receive either 3.5 g⁄d of a prebiotic, 7 g⁄d or placebo. Both doses resulted in improvements in symptoms, with the 7g/d dose being effective for anxiety. Also, both doses significantly increased the beneficial taxa, E. rectale, and bifidobacteria. The prebiotic used here was GOS or galactooligosaccharides. We’ll dive into the individual prebiotics in time, but this is just one of a number of prebiotic options available to drive significant microbiome changes. Through our platform, in looking at the entirety of the microbiome, recommendations would involve several prebiotics in combination to drive the necessary changes. To be clear, we also recommend non-prebiotic products as well; it just depends on the data. Another recommendation could be in regards to diet.

Slide 13 – As we look to this paper, which highlights far more than is within the scope of this presentation, we’ll highlight a few dietary points from a general perspective. As you can see on the left side of the slide, dietary coaching alone can result in a 40-50% improvement in symptoms in depression. When you consider that antidepressant medication is so widely used, but 30–40% of patients do not respond to current drug strategies, one could consider diet as additional therapy. On the right side of the screen, we see a number of very important messengers within this diet and brain health system. Powerful signaling molecules like brain-derived neurotrophic factor, ghrelin, leptin, neuropeptide Y, and more modulate brain functions like appetite, sleep, energy intake, neurogenesis, reward mechanisms, cognitive function, and mood. Like the microbiome, these messengers are largely a function of one’s diet. And generally speaking, negative outcomes occur, in what would be considered the standard American diet, which is low fiber, high animal fat, high simple sugar-based nightmare scenario for health. And when we look at diets specifically, like in a large study with over 87,000 postmenopausal women, we find a progressively higher dietary glycemic index (think standard American diet) was found to be associated with increasing odds for depression. Contrarily, diets high in fiber and vegetables, like the Mediterranean diet, have been shown in studies to be significantly associated with lower odds of depression. So, the purpose of this slide, is to show that not only is diet the biggest influencer of the all-important microbiome, but obviously it goes beyond the simple blood sugars and lipids, but into a complex field of numerous messengers, which interact throughout the entire body, and for purposes of this webinar, to include brain function.

Slide 14 – To return to LPS and intestinal permeability, here we have a slide briefly tackling PTSD. For PTSD in children, there is a fair bit of epidemiological evidence, which reveals that stressful events in childhood, can undoubtedly influence the subsequent onset of psychiatric disorders, such as depression and anxiety. The underlying mechanisms are still unclear, but studies report, that early-life stress leads to a proinflammatory phenotype, as well as alteration of hippocampal neurogenesis, that could have long-lasting negative effects. And a meta-analysis tells us, that the inflammatory markers include not only our aforementioned TNF-alpha and IL-6 but also the commonly measured CRP. So we literally have pro-inflammatory emotional trauma impacting the development of the brain in the young. In adults, we look to two combat studies for more clues.

As you can see on this slide, in a prospective study performed on more than 1800 war zone combatants, plasma CRP levels before deployment were significantly associated with post-deployment PTSD symptom emergence, suggesting that levels may be prospectively associated with resilience versus risk for PTSD. Also, from Bosnian war PTSD sufferers, data identified higher IL-6 levels released by LPS-stimulated blood cells compared with control subjects. This implies stress-induced gut permeability. 

Slide 15 -Let’s continue on the theme of gut permeability, as it is central to much of what we’re discussing here, and in the future. On this slide, we have an interesting study in alcoholism, related to depression and anxiety. In the top right, “CT” stands for control, “AD high IP” represents alcoholics with high intestinal permeability and “AD low IP,” alcoholics with low intestinal permeability. “ADT1” and “ADT2” represent the beginning and end of withdrawal. You can see the significant differences between those with high intestinal permeability, vs. those with low, at both time points, and within each time point, for each category. Interestingly, these researchers found that at the end of the detoxification, the depression and anxiety scores of alcoholism subjects with low permeability recovered completely and returned to the same level as that of controls. However, the alcoholism subjects with high permeability were still characterized by higher levels of depression, anxiety, and craving. Now we know that alcohol is not good for the gut, which includes intestinal permeability, but in this instance, both groups consumed large amounts of alcohol. Therefore, the differences in permeability would be presumed to be from variability within the microbiome, and that’s what was seen when the researchers looked at the fecal microbiome. If you look to the bottom right, you’ll see significant differences between various taxa in the healthy controls, vs the alcoholics with high intestinal permeability. These include many members of the highly beneficial ruminococcaceae family, such as the perpetual good guy faecalibacterium, as well as other good actors like ruminococcus, oscillibacter and something we see more of, the genus subdoligranulum. You’ll notice our generally bad-actor blautia, is highly expressed in the microbiome of those with high intestinal permeability. Once again, there are no probiotics with any of these good genera; we drive these beneficial changes through prebiotics. Here the high intestinal permeability group could benefit, from prebiotics shifting fermentation in the gut, to the much more healthy short-chain fatty acids, as opposed to the less-healthy proteolytic fermentation end products such as phenol, which they found significantly more of in the high permeability group.

Slide 16 – Not surprisingly, faecalibacterium had yet another positive association in this study. Here we have over 1,000 subjects, where the purpose was to analyze the microbiome, as it relates to the quality of life and depression, measured by many different means, as shown across the top right. From this study, we see that when it comes to the quality of life, the two seemingly always beneficial, now familiar, butyrate producers faecalibacterium and coprococcus, are significantly associated with a better outcome. When it came to depression, again coprococcus along with dialister were significantly associated with better outcomes. Although it should be noted that the data on dialister is quite conflicting. With that in mind, we’ll now continue the depression, faecalibacterium and conflicted data thread.

Slide 17 - In this two part study from 2016, the researchers analyzed the gut microbial communities of 58 depressed patients, and 63 healthy controls, which were compared to evaluate whether alterations in the gut microbiome are associated with depression. In the second phase of the trial, they implanted the control and depressed microbiomes into mice. Now I typically will only show you human data, but the second phase of this trial illustrates something that is very commonly seen in microbiome research and something that I think you’ll find to be quite impressive. Here, the mice that received the microbiome from the depressed patients then acted depressed themselves, as shown in the figures to the right, being measured by different parameters. Whether we’re talking about dementia, autism, metabolic syndrome or another condition, time and time again, when the human microbiome from those with a given condition is transplanted into a mouse model, the mice manifest that very same condition. In regards to the first phase of this trial, the findings were generally typical ones, with the healthy subjects having much more of the very healthy genera faecalibacterium, roseburia, and coprococcus. They also had much more of the genus alistipes, which when it came to IBD and IBS seemed to be a beneficial genus, but here when we look at mental health, the data are a bit split.

Slide 18 – So let’s take a little closer look at one of our key themes, the concept of split data. If you recall from our webinars, the genus bifidobacteria is nearly universally considered to be highly beneficial, and for good reasons. However, when we look specifically at active ulcerative colitis, this may not be the case. For a review of this and more, I highly recommend you view our webinar on IBD. Similarly, with the genus lactobacillus, the meta-analyses we have for both IBS and IBD tell us that this genus has far more negative associations in matters of the gut, but for a say in the mode of delivery, breastfeeding and other topics which we’ll soon cover, lactobacillus is a good player. So what we’re looking at, is the labeling of a certain genus as “good” or “bad” may be situational. And this is certainly more true at the genus level, which is where most of our data lay, and as we can dig deeper into each genus, and look at the species level, then some of these variables may be elucidated. As we return to alistipes, on the right side of the slide, we see the overall genus highlighted in yellow, and below that, we see a number of individual species within that genus. So we’re potentially looking at some of these being potential bad actors, and some being potentially good players, or they may simply be associated with a certain environment. Given that two studies, including this one here, show increased alistipes in depression, in addition to pain and a fibromyalgia study, this genus you’ve probably never heard of before, warrants a further look from us now, and in the future. And in fact, we do know that these species are able to degrade tryptophan, thereby potentially reducing its availability for serotonin synthesis. In addition to illustrating the situationally good or bad aspect, this illustrates another important point in microbiome research. As much as we know, and it’s quite a bit at this point, we don’t have all of the answers. What we’re doing is tip of the spear stuff here, and as we generate more data points with our cutting edge shotgun metagenomic analysis, we’ll be less encumbered by the limitations of the old data, and possibly uncover at the species and situational levels what’s going on, like in the case of alistipes and its potential role in tryptophan. 

Slide 19 – To continue on the theme of tryptophan, as you can gather from this webinar, depression, and anxiety are not all about serotonin. Although the pharmaceutical approach to both relies quite heavily on modulating serotonin availability, I hope you can appreciate that there is much more to the equation. This is not to say that the role of serotonin should be minimized. With that said, let’s consider the mast cell once again. They not only secrete histamine but a wide variety of other compounds as well, to include serotonin. That’s right; serotonin is a part of the inflammation process. Which is intriguing, as we know some 90% or more of our bodies’ serotonin is found within and around the gut. This neurotransmitter is a bit of a double-edged sword, a topic we’ll in time, dive much deeper into in its own webinar. But for now, if we just consider the theory, that in the chronically inflamed gut, we have an exhaustive turn-over of serotonin pools. Now consider that serotonin is derived from the amino acid tryptophan, and that tryptophan is the least occurring amino acid in the diet, at about 1%. Now also consider, that although a person with an inflamed gut may be producing serotonin, and although it makes its way into circulation, it cannot cross the blood-brain barrier. So what we’re looking at, is the exhaustive inflammatory production of serotonin in the gut reduces the availability of tryptophan for the brain. And serotonin is key when it comes to mood, as we can see through the emphasis on the SSRIs. And in fact, we see that in the literature, that depression is associated with reduced plasma tryptophan. So a key factor here is to reduce the level of gut and systemic inflammation, stress, and toxic compound production. 

Slide 20 – And speaking of gut-microbiome derived molecules, here we have a great table which lists many species and strains which are capable of producing the respective neurotransmitters in the left column. As you can see, it contains both good bugs and opportunistic pathogens, capable of producing the exact same neurotransmitter. Notice the number of lactobacilli capable of producing histamine. We typically associate histamine with mast cell reactions, but here we have a list of taxa capable of producing it on their own, within the lumen of the gut. Whether the histamine comes from mast cells or these taxa, it is still a histamine and has undesirable effects in excess. And even though this paper was published in 2018, just a casual glance shows us it’s not all-inclusive. For example, from our first webinar, if you recall there were two species, you should be concerned about if you take L-dopa for Parkinson’s. They are Enterococcus faecalis and faecium. Both can take the L-dopa and convert it into dopamine, which is then unable to cross the blood-brain barrier. Neither is listed here. Also not listed is lactobacillus reuteri, yet another species from lactobacillus capable of producing histamine. 

Slide 21 – I hope this webinar gives you an appreciation for the many factors at play when it comes to the health of your gut and your mental wellbeing. As I try to keep these webinars at a reasonable time limit, I’m unable to tackle every possible aspect within this very broad topic. My hope is that you walk away with a few new pearls of wisdom, in order to broaden your understanding of the complexity of the body, as it related to the microbiome. I think by now, it’s becoming clear that a healthy microbiome, which is well-populated with a diverse array of many of the beneficial bacteria which keep coming up like faecalibacterium, coprococcus, roseburia, prevotella, bifidobacteria and more, lends itself to a state of health, and avoidance of inflammation. In the webinars to follow, you’ll see many of the same bacteria listed in their familiar roles, as you’ve seen in the webinars we’ve completed. A key concept I want you to walk away with is this. At its essence, there is no dementia bacteria, no Crohn’s or ulcerative colitis bacteria, no depression or anxiety bacteria, but in disease, a dysbiosis of good actors vs. ones whose end product is inflammation, and so one person’s autoimmune disease, is another’s dementia, is another’s depression. It’s the amazingly complex interplay of the microbiome, your genes, luck, and lifestyle factors which largely inexplicably result in a roll of the dice. Better to cover your bases, and take a peek into your microbiome with the help of those who understand it as well as any other. 

In our next webinar, we’ll dive into the relationship between metabolic syndrome and the microbiome. So for all of you out there concerned about your, or a loved one's lipids, blood sugars, weight, and a host of other metabolic parameters, stay tuned. Until next time.