[gentle music] - Margaret Alexander: Good evening, everyone, and welcome to our Crossroads of Ideas, Gut Instincts: Microbes, Addiction, and Immunity, part one in our microbiome miniseries.

I am Dr. Margaret Alexander.

I am an assistant professor here at UW-Madison in the Department of Medical Microbiology and Immunology.

I did my post-doctoral training at University of California, San Francisco and my doctorate at the University of Utah.

Today, we're gonna be talking about this Crossroads of Ideas.

And this is a collaboration between the Wisconsin Institute for Discovery, the Morgridge Institute of Research, and the UW-Madison Strategic Communications.

And it's an opportunity to foster dialogue between community and UW-Madison researchers.

The series addresses issues that matter to all of us, and are also the subject of research that is going on at UW-Madison.

Since 2014, Crossroads has been a staple of public programming and offered at the Discovery Building at UW-Madison campus.

So tonight, we're here to kick off an exciting three-part miniseries on the gut-brain superhighway and the complex interplay between microbes, addiction, and immunity.

I'm going to give a brief introduction of my connection to this topic, and then I will invite my colleague, Dr. Vanessa Sperandio, to the stage for an overview of her work and then a brief conversation with me, and we'll make sure to save time at the end for audience questions and answers.

So please have your questions ready for us.

All right, so today, we're here to talk about Gut Instincts: Microbes, Addiction, and Immunity.

And my research focuses on two of these, in particular, these microbes and the immunity connection that we're talking about today.

But before I go into my research, I'd like to share this poem excerpt from a Sylvia Plath poem called "Mushrooms" because for me, this really sort of gives an image of our microbiota.

So here's this excerpt.

"Nobody sees us, stops us, betrays us.

"Small grains make room... "We diet on water, on crumbs of shadow, "bland-mannered, asking little or nothing.

"So many of us!

So many of us!"

And I love sharing this poem because it really brings up this image of the microbiota, which are these trillions of microorganisms that are colonizing both inside of us and our gastrointestinal tract, and on us, on our skin.

And these microbes consist of bacteria, of viruses, and of fungi, and are known to have significant impacts on our health as well as disease.

And so the research that I work on and our lab works on is focused on thinking about the mechanisms of the interactions between our diet, our microbiota, and our immune system.

And we're particularly interested in this in the context of autoimmune diseases.

And the way we approach this, we come at this from a couple different angles.

One of which is a microbe-centric approach, where we sort of take the perspective of a microbe and think about microbes that are altered in disease states like autoimmunity, and try and understand how those microbes impact immune responses that are contributing to these autoimmune disease states.

And so, with this microbe-centric approach, you might think of a microbe that is elevated in an autoimmune condition like multiple sclerosis.

So our questions are, is that elevation leading to changes in immune responses that contribute to disease, and can by understanding more about how these microbes impact immune responses, can we target interventions for those pathways, that cross talk between microbe and immune response?

And one of the ways we can target this is thinking about diets that might modulate the conversation between our microbes and the immune response.

So I won't talk too much about this today.

I just want to give you a brief thought about thinking from the microbe perspective.

But another way we approach this question is thinking about it from a diet-centric approach.

So this, we think about diets that are used in the clinic or in clinical trials to mitigate disease in autoimmunity.

And what we really want to understand is how these complex diets impact these autoimmune disease states.

And trying to understand the interactions between microbiota and immune response that happens during this that can mediate that response.

And so this is a big question of how do diets impact autoimmune diseases.

This is a pretty intimidating question to look at at this scale.

And so what we do is try and focus on specific examples.

And so the example I'm gonna tell you about today is about a ketogenic diet and multiple sclerosis.

And this was really exciting and interesting to us because there have been now phase two clinical trials, as well as many mouse models of multiple sclerosis that have seen a protective effect of a ketogenic diet in multiple sclerosis.

But the big question was, how exactly is this happening?

Can we figure out more about the host and microbiota mechanisms that are driving this, so that we can understand more about why there might be variation in patient responsiveness to this diet?

Can we get better specific bioactive compounds that might be sufficient to mediate these things without having this complex diet?

And that is because, as I was saying, ketogenic diets are complex.

They're sort of defined by the strict restriction in carbohydrates.

And that's really shifting host metabolism to instead of breaking down carbohydrates as an energy source, breaking down free fatty acids.

And this process is called ketogenesis and actually is what the ketogenic diet is named after.

And so what we wanted to do is try and understand more about the mechanisms of how this protection takes place.

And so I'm gonna walk you through sort of, we just published this story recently thinking about how a ketogenic diet is protective in mouse models of multiple sclerosis.

And we sort of started this with our thought that a ketogenic diet is complex.

And so are there things that are changing in the host during a ketogenic diet that might be mediating this protect without having to have the complex diet?

And one of the things that I mentioned happens is this switch to ketogenesis, where you're making ketone bodies as a form of energy.

And so what we wanted to do is see, can we pharmacologically elevate levels of ketone bodies like beta hydroxybutyrate and get levels of protection that are similar to that complex ketogenic diet?

And that was one of the things we observed.

Just by increasing levels of beta hydroxybutyrate, we were able to get protection in our mouse models of disease.

And so this gave us some insight on the host mechanisms that were happening in this protection.

But we wanted to know what was happening with the microbiota, because there's a strong link to the microbiota and mediating autoimmune disease phenotypes.

And so one way to get at this is to compare response to diet in mice that have an intact microbiota versus mice that don't have any microbes at all, which are called germ-free mice.

And when we compared germ-free mice to normally colonized mice with intact microbiota, the germ-free mice didn't have a protective effect in response to a ketogenic diet in our models of MS. Really telling us that the microbiota is necessary to be there to get a benefit from that diet in the model that we're using.

And then additionally, what we wondered is, is a microbial community that's shaped by exposure to this ketone body, beta hydroxybutyrate, is that gonna be sufficient to give protection from disease?

So can we take microbiota that's shaped by the presence of beta hydroxybutyrate, put that into a mouse, and see if that looks the same as just giving the supplemented beta hydroxybutyrate?

And that is what we saw.

A community that's shaped by BHB was also able to convey that effect.

And so we have this idea that the ketone body, beta hydroxybutyrate, shapes the composition or function of the microbiota in some way that is mediating some of these protective effects.

And then our next questions were, okay, the microbiota, as I was saying, there's trillions of microorganisms.

There's hundreds of species.

So are there particular species that are mediating this?

And so we looked into this and used some previous literature and data out there in the field to kind of get at what members of the microbiota might be involved in this.

And what we saw is that there was a member of the microbiota called Lactobacillus that was able to be increased by exposure to beta hydroxybutyrate.

And so we wondered if just by supplementing mice with Lactobacillus, could we get similar response.

And then additionally, what we knew from the literature in the field was that Lactobacillus make this metabolite called indole lactate acid.

And indole lactic acid had been seen to have this potent immune effect where it can inhibit immune responses that are known to contribute to multiple sclerosis pathologies.

And so what we did is just by supplementing mice with our Lactobacillus or this metabolite indole lactate, we saw a similar amount of protection, which we think is acting through mediating signals to the immune responses that contribute to multiple sclerosis phenotypes.

And the specific response that we were looking at was these adaptive immune responses called T helper 17 responses.

And so this is the story so far that we think is happening in this protective effect of a ketogenic diet and multiple sclerosis, where the host is making beta hydroxybutyrate, impacting the microbiota and production of metabolites that can then feed into immune responses and inhibit immune responses that are contributing to multiple sclerosis.

But we have a lot of things that we're needing to do to figure out more about these connections.

So how exactly is BHB impacting the microbiota?

How does this microbial metabolite impact the immune response?

And additionally, are there other things that are happening during a ketogenic diet that might also lead to some of these effects?

And these are some of the things that we're currently working on in the lab.

And so with that, I want to acknowledge everyone that helped do this work.

And thank you all so much for your attention.

And I will transition now to Dr. Sperandio.

I would like to introduce my colleague, Dr. Vanessa Sperandio.

Dr. Sperandio is the chair of the Department of Medical Microbiology and Immunology here at UW-Madison and the Robert Turell Professor.

She was the Jane and Bud Smith Distinguished Chair in Medicine and a professor in the Departments of Microbiology and Biochemistry at UT Southwestern Medical Center, and she received her bachelor's in biology, her master's and PhD in molecular genetics in the State University of Campinas in Brazil.

Please join me in welcoming Doctor Sperandio.

[applause] - So I know I have a very long title, but the gist of this talk is chemical interactions between host neurotransmitters, microbial chemical signals that they make with pathogens, the microbiota in the intestine and the host in health and disease states.

So the gut-brain axis is the two-way street of communication between microbes and the host.

There is a strong correlation between microbiota composition and microbial pathogenesis with host neurological diseases and behaviors.

Members of the microbiota are known to modulate the levels as well as the activity of neurotransmitters in the gut.

And this rich chemical exchange affects bacterial pathogenesis, amongst other processes.

And we want to exploit this chemical signaling and understand it so we can develop novel types of therapies for diseases.

So I'm gonna focus on several of neurotransmitters that are found in the gut.

And I'll start in why do we look at neurotransmitters in the gut?

Because your brain is connected to the gut and you have a highly innervated enteric nervous system.

That's why you have that "gut feeling."

And you're basically, your lumen here, where the microbiota is and where pathogens come first, is full of a whole array of neurotransmitters that microbes see every day.

So they learn to sense them.

So mammalian cells tend to sense this neurotransmitter using these types of receptors here that are called GPCRs.

However, bacteria usually don't use that.

Their preference is to use receptors in their surface that are called histidine kinase.

What happens here is these receptors, upon binding to their signals, they will phosphorylate themselves.

That starts a signaling cascade in the cell.

That goes on to change gene expression within a bacterial cell.

So all of these neurotransmitters that are sensed by bacteria, they cross react with bacterial signals too, okay?

And this cross signaling here, we call it interkingdom signaling because as I mentioned, mammalian cells use hormones and neurotransmitters to communicate with each other and coordinate behaviors.

Bacterial communities also use chemicals that in the field, we call autoinducers, in a process known as quorum sensing to coordinate population behavior amongst bacterial cells.

So the first system that we studied in my lab is this one, in which the host neurotransmitters are epinephrine, norepinephrine.

This is adrenaline and noradrenaline.

You can say bacteria can be adrenaline junkies too.

And they are stress neurotransmitters involved in the fight or flight.

But importantly, they control intestinal motility, chloride and potassium secretion, and also impact immune responses in the host.

The bacterial signal that cross talks with this one is this signal here that we call autoinducer-3.

For chemical aficionados, these are pyrazinones.

They are made by many, many species of bacteria, but importantly, they're made by the microbiota in the gut of human beings and also mice.

The way this system works is you have one of these histidine kinases.

In the case, we named this QseC.

And it will sense either the epinephrine/norepinephrine from the host or the AI-3 from the microbiota.

That activates the kinase.

So here is no activation, no, just basal phosphorylation.

You can see that just by how big these bars are.

Here you have the bacterial signal AI-3.

Here you have epinephrine.

And both of them, it activates all the way.

And what this translates into the signaling cascade is activation of virulence in many bacterial pathogens.

All of the virulence programs.

Just for you to have an idea, this system controls pathogenesis in gastrointestinal pathogens, urinary tract infections, ventilator associated pathogens, and many others.

So it has always been thought of as a good target for antimicrobial therapy.

And yes, QseC, which is the bacterial sensor for this can be inhibited using alpha-adrenergic antagonists such as phentolamine.

But they also will hit mammalian adrenergic receptors, which is not desirable.

So we did chemical screens to look for molecules such as this one, LED209, which selectively will hit the bacterial QseC but will leave the mammalian adrenergic receptors alone.

And by using this, we've been able to prevent virulence in vitro and during mammalian infection of many, many bacterial pathogens.

And the reason, and these strategies are what we call anti-virulence approaches, because you're inhibiting the start of the pathogenesis process in the bacteria, but not growth, and you're not killing them.

And the thought process in that is that there is less selective pressure for this bacteria to develop antimicrobial resistance because you're not going with a hammer.

You're going with a scalpel.

So, but are all stress, but all signaling in this interkingdom communications, is it always stressful?

And the answer is not necessarily because you do have several neurotransmitters in your gut that are kind of calming, endocannabinoids being one of them.

And yes, you have endocannabinoid receptors in your gut, okay, and that sense plant cannabinoids.

But you didn't evolve to sense the plant cannabinoids.

The reason you have these receptors is because you yourself in the intestine make plenty of endocannabinoids that you make yourself.

And the one we work with is 2-arachidonoylglycerol, 2-AG.

And don't worry, not a thing.

Everything's gonna be all right.

But please don't get out of here and go do something and tell people that I told you to do so because I did not.

[audience laughing] So what does this endocannabinoid system does for your physiology?

As I mentioned, this is a common thing.

It decreases inflammation.

It reduces colitis susceptibility.

And in fact, recreational cannabinoids have been used to alleviate IBD symptoms and also colitis.

And the one that we study is 2-AG.

And it also interfaces with the microbiota because there is one member of the microbiota, this is species here.

If you care, it's named Akkermansia muciniphila .

It increases the levels of endocannabinoids that you make in your intestine.

So there is a cross talk in there.

And how does this work?

I told you, epinephrine/norepinephrine are stress neurotransmitters, okay?

They stress you.

Endocannabinoids are calming, and they act through the same receptor as epinephrine/norepinephrine in the bacterial cells.

I told you, epinephrine/norepinephrine will activate the QseC to start a virulence program in many bacterial pathogens.

Endocannabinoids, such as 2-AG, will prevent this from being activated.

It will actually inhibit it.

And that will stop the virulence programs.

So they are antagonistic to each other even though they work through the same bacterial receptor.

And what's interesting is that in mammalian systems, epinephrine/norepinephrine and endocannabinoid signal in the intestine, they have opposing functions.

Epinephrine/norepinephrine increase intestinal motility, inflammation, and stress.

And endocannabinoids decrease motility, inflammation, and of course, it decreases the stress too.

And how can we think about this in a therapeutic way?

Okay, you have plant cannabinoids.

One of them, cannabidiol CBD, seems to be, according to the press, the cure for everything.

But it is used sometimes to alleviate colitis.

And the beauty of CBD is it is FDA approved and considered safe to be even, to be used even in children that are two years of age or older.

So I'm gonna go to the last system that we work with on this interkingdom interactions, and that's also a calming one.

This one involves serotonin, your feel-good neurotransmitter, and a bacterial signal that Maggie alluded to, indole, that's made by the human microbiota in the gut.

Okay.

The way this works is, first of all, serotonin makes you feel good, but 95% of that is made in your gut.

And the way this works is there is the sensor kinase.

This one has a different name, a different receptor.

It's called CpxA.

This is usually activating bacterial virulence.

However, when serotonin or indole binds to this kinase, what they do is they shut it down.

And when they shut it down, they shut down virulence in bacterial pathogens.

So it's opposing functions to the adrenergic signaling that I just told you about.

Similar to the endocannabinoid one, but working through a different receptor.

And why do we care?

First of all, serotonin is something that people take.

And serotonin agonists can actually alleviate enteric disease.

You can use drugs that are used for something else and probably adapt them to use for treating infectious diseases.

Also, Prozac inhibits serotonin reuptake from your gut.

So what happens is when you take Prozac, you accumulate serotonin in the gut, and at least mice in these conditions become less susceptible to enteric infections by bad pathogens such as E. coli.

If you like Quarter Pounders from McDonald's, you probably know what I'm talking about, salmonella and so forth and so on.

And finally, this does not just play a role in infectious diseases, but it also plays a role in addiction behaviors.

So what we have been studying in the lab for a while now is that people who are addicted to cocaine.

So when you give cocaine to mice, because we do everything in mice, okay, what happens is cocaine is a psychoactive drug because it makes you accumulate norepinephrine.

So you keep firing.

And that's why people are always excited when they are on cocaine.

And when that happens, norepinephrine, as I mentioned, is sensed by the sensor here in bacterial cells.

And E. coli, part of your commensal microbiota, has those.

And when this happens here, you have cocaine.

A lot of norepinephrine is released.

It's sensed by bacteria in the gut.

And E. coli expands, it blooms.

Usually you have very few E. coli.

Now you have a lot of E. coli.

And when you have a lot of E. coli, E. coli likes to use glycine, which is an amino acid, as a nitrogen source, as a food source.

And that causes the glycine levels to drop in the gut.

And they also will drop in the brain.

And glycine is actually in the brain, a ligand, okay, that activates the systems that prevents you from being addicted to psychoactive drugs like cocaine.

So when you have this situation here with a lot of E. coli, drops in glycine, this doesn't activate.

There is increased cocaine addiction behaviors.

Just for you to have an idea of what I'm talking about, we give cocaine to mice with and without E. coli.

So, and look at them, it's labeled.

But I don't think I need to tell you which animals are on cocaine here, right?

It's pretty obvious.

[audience laughing] So it's fun to see mice on cocaine running around, but as a scientist, need some numbers.

So what we do, we have these boxes and they have lasers in them.

So when the animals run around, they cut the lasers.

And then that tells the computer when they cut them.

And that measures how much they run and how they run.

And then you can get graphs that we scientists like.

Okay, so here we don't care.

These mice never got any cocaine.

They're boring, okay?

Now, if you have mice that have a microbiota that has no E. coli in it, they respond to cocaine.

This means that they're running around more, okay?

But it's a regular response.

However, here in blue and in green, if they have a lot of E. coli in their microbiota now, they have enhanced behaviors and they run like crazy.

So in summary here, cocaine exposure increases norepinephrine.

The gut that leads to a Proteobacteria, an E. coli bloom.

Proteobacteria used glycine as a nitrogen source, decreasing its levels in the gut and in the brain.

That prevents glycine from activating the signaling system that prevents addiction.

So what it ends up doing is that you get these animals to become more susceptible to cocaine.

And you can fix this by giving glycine back to mice, okay?

And we wonder now what other members of the microbiota do to sensitization to drugs such as cocaine.

We know what E. coli does.

We don't know what the other ones do.

There is a lot of wealth of microbes in there, and it opens the door for the manipulation of intestinal bacterial processes to be therapeutic targets to impact the course of substance use disorders.

So what I told you today is that the gut has a wealth of neurotransmitters in them.

And microbes, both members of the microbiota and also pathogens in the gut are exposed to them.

They have sensors for these neurotransmitters, and usually the sensing is tied with interkingdom signaling, sensing of other bacterial signals, too, because they're chemically structured very similar to each other.

So they kind of learn how to see each other.

And manipulation of the signaling systems can lead to novel therapeutic strategies, not just for infectious diseases, but also to treat substance use disorders.

And I'll finish here with the people who actually do the job, which are the young people in the lab who work very passionately about, towards their science to get with all of these ideas together.

So, thank you.

[audience applauding] - Margaret: All right, So I'm gonna pepper Vanessa with some questions.

And we'll have a little bit of a discussion.

And then we'll open it up for some Q&A from the audience.

Vanessa, I always have this question I feel like when I hear the cocaine story.

Which is, you know, thinking about maybe coming at this from a, you know, a microbe's perspective, where if you're E. coli and you're expanding when you're exposed to cocaine, and then you're altering the host's behavior to enforce that addiction, is this just a giant ploy of the microbe for increasing its fitness by altering host behavior?

And do you know of any other examples of that?

- Vanessa: So they're not too many examples that got to the mechanism of things.

- Margaret: Mm-hmm.

- Okay, but there are very complex interactions between composition of the microbiota and expansion of certain members with opioid addiction.

They don't, they didn't get all the way to the mechanism and the ins and outs of that.

But it has been looked at, at opioid addiction, alcohol addiction.

- Margaret: Yeah.

- And, um...

I'm not sure if they actually looked to much of the microbiota in people who smoke a lot of marijuana, but at least with opioids they did.

And with psychoactive drugs like cocaine, there is.

- Margaret: Gotcha.

And I think, remind me too of the mechanism of the glycine.

So E. coli is decreasing glycine.

And then how is that feeding into the behavioral change?

- So the way this happens is E. coli eats the glycine, right?

- Yeah.

- Your cells want glycine, so.

That decreases the level in the gut.

That goes systemically, decreases the level of glycine in the whole body, including in the brain.

And glycine is actually a ligand for this receptor in the brain, for a pathway that controls addiction.

Not only addiction, but also schizophrenia.

Because apparently it's involved in both.

And when that signaling doesn't happen, you get less protected against developing an addiction behavior.

- Margaret: Gotcha, super, super fascinating.

I guess I think of this, too, in the context of, you know, microbes influencing host behavior with diet, and this, I, you know, I don't think there's that much out there, but one of the, like, sort of out there ideas that I have been wondering about is if, you know, a diet can sort of shape a host's behavior in terms of dietary preferences.

There's actually this paper recently that was a knockout of a fatty acid sensor molecule, and they saw a change in the microbiota and then a change in, like, the sugar preferences of mice based on that.

And it's always interesting to me to think about that if there's, you know, some sort of connection back to, you know, the fitness of the microbes based on those sugar preferences.

And I don't remember if they got around to that or not, but it's something.

- Vanessa: So there is a tie-in with diet.

I didn't get time to go to that, but I mentioned briefly that you can fix this whole problem just by giving glycine to these animals.

You can correct that.

And it turns out that glycine is made out of this compound called sarcosine.

It's the direct precursor.

And you can fix the behavior too if you give sarcosine.

And sarcosine, for anybody who goes to the gym a lot or work out a lot, or athlete, is used very commonly as a dietary supplement by athletes.

You can go to GNC and buy sarcosine.

And that basically immediately turns into glycine and fixes the behavior.

So that there is some tie-in with diet.

And this is obviously in the very beginning.

So we have a lot of ground to cover.

- Margaret: Definitely.

I think one of the questions I've always wanted to talk to you about is sort of, do you have any out-there ideas in terms of microbiota, you know, gut-immune-brain axis that you've wanted to look into and haven't had the chance to do?

- Vanessa: Yes, because there is a very strong, people are seeing a lot of correlations between inflammation and different behaviors.

You know, you have things like that happening with autism.

Autism, I think, is the one that people look the most at.

Alzheimer's, right?

In our case here, it doesn't seem to be inflammation.

We did look at inflammation.

It wasn't the inflammatory process, but... - Margaret: So sorry.

- Which was what we thought was gonna happen first... [both laugh] ...to be quite honest with you.

But with other neurodegenerative diseases, the connection between inflammation, especially with dysbiosis, changes in the microbiota to get a microbiota that's pro-inflammatory.

- Margaret: Yeah.

- Okay.

That tends to affect a lot of behaviors.

There's also a lot of work coming out on depression.

- Margaret: Yeah, 'cause that's something I think a lot about too, is we haven't sort of gone into this realm, but there's, you know, the immune cells that we're looking at in multiple sclerosis.

There are some ideas that these Th17 cells can have an impact in depression.

And if we, you know, modulate that interaction between the microbiota and those cells with diet, you know, can you try and, you know?

- Vanessa: Yeah, you probably can.

Because as you know, when you change the diet, you change the composition, right?

So if it's a plant-based diet or a ketogenic diet, or, or a Western diet that's you know, full of carbo-- processed carbohydrates, you have very different microbiotas.

And, unfortunately, the Western diet seems to be the most pro-inflammatory one.

- Margaret: But my problem too, is, you know, trying to study, you know, behavioral conditions like depression, it's, how do you model that in mice?

Because as you probably both appreciate it, we're both working in mice.

And so how do you assess if a mouse is depressed?

[Vanessa laughing] I don't know the answer.

I know people are working on that, but I think it's an interesting... - There are mouse models, okay?

The same way there are mouse models for autism.

- Margaret: Mm-hmm.

- Vanessa: Okay?

But I'm not a neuroscientist, okay, I'm a microbiologist.

But what a neuroscientist is gonna tell you is that you cannot rely on just one model.

You have to use many of them.

I showed you, for example, for the addiction studies that we did, I showed you just locomotion, but that's, like, the starter model.

Okay, the easy one.

Then you have drug-seeking behavior on, like, do they seek the drug more.

And the answer, yes.

And there are models for that, like, different types of boxes, okay, where they go.

You just give drug to them in one type of box.

Normally, they would spend the same time in both.

But do they develop a preference now to the one where they get the drug?

And there's also self-administration, which it's easier to do with rats because they're bigger and they're, they're way smarter than mice, so... [laughing] They are.

So you can train them much better.

With mice, usually you just train about 50% of them to press the lever so then you can do the behaviors.

With rats, you can actually train many of those.

And, of course, extremely expensive and very, very difficult to do would be with monkeys, with primates.

- Margaret: So I think you had an example today of sort of how signals from the microbiota get sensed in the brain, which is actually really interesting to me because I, you know, I think about, you know, how, what are the ways in which changes in your gut can influence the brain.

And I was wondering if you, you had any other examples of sort of this, this, you know, the signals being sensed in the brain.

One of the things I think about is, is it the signal that's reaching the brain, or are there changes happening, sort of the immune response in the gut that then traffic to the brain and trying to figure out which one of those things might be.

- Vanessa: So we have something very preliminary on that, okay?

And that's with indole, which is strictly a microbial signal made in the gut, not in the brain.

- Margaret: Yeah.

- And if you do metabolomics in the brain of mice, okay, you can actually find indole in there.

So some of-- - Margaret: How much indole, though?

- Ah.

That, with cerebrospinal fluid from mice, is hard to measure.

[both laughing] But-- - Margaret: I'll ask you in a year.

- Yeah, but you can actually find indole in there, so somehow it travels.

I'm assuming it goes through the, through the blood, because with the whole thing with glycine levels.

- Margaret: Yeah.

- It's through the blood.

It's systemic.

So I'm assuming that it's just going systemic through the blood.

- Which can happen, I guess, for these small molecules.

I mean, you guys saw the indole is only less than 12 carbons, I guess.

I can't remember.

I'm trying to map it out in my-- But, you know, a very small thing.

And so I'm wondering if there's, you know, bigger things that it's more difficult to reach the brain than you have to have things that change, you know, maybe to the enteric nervous system that then feed into the brain as another way that those things that quite can't get systemic can influence the... - Yeah, I'm not sure if, like, big fatty acids would make it.

Right?

- Yeah, right?

- Those I think would have some trouble going there.

- Margaret: I would think so too, but... - But a lot of the signals are very small, and some of them are modified versions of amino acids or catecholamines, which are also pretty tiny.

So it all depends on the type of signal that you have.

- And I guess maybe I'll ask one final question of thinking about, like, how reversible these things are.

So this, you know, cocaine increasing E. coli levels that are sort of feeding into the cycle.

How reversible is that?

If you stop this and still have that increase of E. coli and you sort of let it, the mice try and equilibrate for a couple of weeks.

Do they still have that addiction, or can you sort of get to a point where that no longer is the case?

- Amazingly enough, whatever happens in there seems to have a long-lasting effect.

So my former postdoc, Santiago Cuesta, who did the studies then is continuing those in his laboratory at Rutgers University, he actually looked at relapse.

- Margaret: Mm-hmm.

- And of course, after you stop and give it a long time without the drug, the microbiota gets to normal with everything again and you wait enough, what would be long enough for a mouse to expose them to cocaine again, and they relapse much more than the ones who never got this process to begin with.

- Margaret: Yeah.

- Why?

We don't know.

But whatever modification is happening, they relapse too.

- Margaret: Very, very interesting.

Well, I want to now open it up to the audience for Q&A.

If you would raise your hand, we'll have people come around with microphones so that people online can hear as well.

So any questions?

- Attendee 1: Hi, thank you.

Those were two great presentations.

I have a question for you.

[chuckling] Um, yeah.

[chuckling] About your work with the autoimmune system in your mouse model.

So where does-- Since these mice are in a very controlled environment, where does the microbiome for these mice come from?

- Margaret: It's a great question.

So in our mouse facilities, there's a couple of different levels of definitions of microbial communities, I'll say.

So one is germ-free, so no microbes.

And the other is specific pathogen-free, which is, they're sort of tracked for different pathogens.

And we want to exclude those pathogens because we're trying to have a mouse colony that is not constantly undergoing infection.

That influences our immune responses, that confuses all of our experimental systems.

And that's the sort of system that we work.

So it's not sort of, it's a non-diseased, I guess, or non-infection microbes.

Now, this is a really interesting question because there's sort of different levels of this.

So you can screen for many pathogens.

You can screen for a subset of pathogens.

And so there's different levels of that specific pathogen free.

And the other sort of side of this is more dirty mice, which are actually termed dirty mice because it's, like, some people use pet store mice or mice that are captured in the wild and try and colonize, you know, these mice that we use in the lab with those microbial communities to try and better model, you know, what we're all experiencing, which we obviously are exposed to pathogens in our lives.

And so, how does that impact this, you know, cross talk between microbes and immune system, if you've had this sort of exposure to pathogens.

And this is actually something really interesting to think about too, when you're thinking about immune responses, you know, how does that differ based on if you've been exposed to pathogens or not?

So I kind of went off on a tangent there, but I think it's a really interesting question of, sort of what are these background microbiotas.

And it might change your response very differently depending on what that background microbiota is.

So let's say I have a microbiota and my twin, which I don't have, we'll pretend my twin has a different microbiota.

We both eat the same diet.

We might have different responses to that diet based on the background microbiota that existed in us.

Yeah.

Great question.

- Attendee 2: I had a question regarding how the diet influences autoimmune diseases.

And based on your diagram, you had BHB and then that influences the microbiome, and then that triggers this small molecule.

And how do you identify, like, these different types of checkpoints and, like, are they already known prior to, like, formulating your research questions and, like, what challenges you have, if maybe those checkpoints and that map, like the figure you showed, aren't necessarily so clearly defined?

'Cause I know you're also doing this in mice and it's a lot of moving parts.

So it's kind of a broad question.

- Margaret: Yeah, it's sort of like, how do we formulate the questions that we are trying to answer, of like, why did we initially look at, you know, beta hydroxybutyrate and ketogenesis, which was all based on, you know, as, as people say, we build on the backs of giants, right?

We know all of this, all of these changes that happen in response to this diet on the host side.

And so can we use that previous information to sort of think about, okay, what is a likely way in which this is mediating a response in the context that we're looking at?

And so, that's one thing that we can do is sort of take advantage of all of the work that has done, been done before to come up with these more targeted and informed hypotheses to then go test.

In addition, what is known in the literature is that the diet can profoundly impact the composition and the function of the microbiota.

And so that's a big thing to know.

Thinking about why did we look at the microbiota in the first place?

That is something that sort of builds on that.

But then too, thinking about the specific microbes that we go after and those metabolites, that is also looking into, you know, what has been done previously, where people had seen that the Lactobacillus can have a protective effect in a mouse model of multiple sclerosis.

And so using that information, we can say, "Oh, we saw an increase in Lactobacillus.

Maybe that is also explaining our phenotypes."

And then we can sort of build off of that and see what else is known about those interactions in the literature to get at those bioactive microbial metabolites to test those as well.

So it's trying to synthesize all of this, you know, background information and string it together to sort of make sense of this bigger-picture question of how does a diet impact a autoimmune condition?

Yeah.

- Attendee 3: One that I was definitely thinking about, and I mean, in a way, like we talk about like, "Oh, yeah, this was done in a lab "with, like, controlled mice.

And then how does this translate into people?"

I mean, one of the things that, like, your experimental procedure about, like, oh, you have these like, mice with no microbiome.

It feels a lot in some ways to, like, if you're about to go get surgery and you have to take, like, a broad-spectrum antibiotic for a long time specifically to prevent, you know, other types of infections.

But now maybe you've totally cleared the board on your own microbiome.

Is, so in that situation, is there, like, a, whether this exists or maybe something that you would like to dream up, like, if you had a chance to build your own best microbiome, like, boo, E coli.

Yay, Lactobacillus.

[Vanessa laughing] Like, are there things that, like, practices like that today or, like, things that are working towards, like, clinical applications like that?

- Vanessa: Well, I can take this one.

So actually...

There is treatments like that, and people are looking very seriously into that for people who get infected with Clostridium difficile , okay?

Especially if you go through broad spectrum antibiotics for a very long time, Clostridium takes over and it can cause necrosis.

And if you keep trying to kill it with antibiotics, you just make things worse because it doesn't repopulate.

So with all of the other species that control that one and keep it in check.

So the really efficient treatment is stool replacement.

So I'm not making that up.

So you have, it can either go through an enema, or they have capsules that you ingest.

And they get basically stool donation from very healthy donors to repopulate your intestine with a good microbiota that's gonna get rid of that pathogen for you.

And of course, nobody wants to hear about stool replacements, especially patients.

So there is a lot of work being done on trying to figure out which are the members of this microbiota that can actually take care of business and try to make synthetic microbiotas with these specific members.

So you would have something more defined, especially because, you know, FDA is not a huge fan of, like, just giving stool from one person to another, but.

- Margaret: And how do you define healthy, too, right?

- Vanessa: Yes, and how do you define healthy.

- Margaret: Yeah.

- Vanessa: So in desperate, it's a desperate measure, and it's one that's worked, and it is used.

But the effort in science is towards defining what would be your best microbiota to get rid of this.

- Margaret: And too, I'll just add on a little bit to that of like, you know, how do you define that for, for this case of C. difficile infection, it might be different than the case of an autoimmune condition.

And so your optimal synthetic microbiota or even microbial metabolites is going to differ between, you know, what context you're trying to deliver that therapy in.

And so trying to figure out exactly how these things are working is gonna help us, you know, get that best community for that exact context, yeah.

- Attendee 4: I just wondered if both of you could answer this question about what do you see on the horizon as far as ways that your research could go to develop this further?

- Vanessa: So in our case, one of the things that people are not, which is going to be the next crisis we're gonna have, okay, it's antimicrobial resistance to antibiotics.

And pretty much, there is resistance to everything there is in the market, okay.

Because they develop resistance really, really fast.

And we have to find new ways to develop them and trying to adopt drugs.

And it's not just me, but a lot of people work with the microbiome, they're looking into screening FDA-approved drugs to see if they can be used in the context in which they can control bacterial infections, because people tend to think about antibiotics like, "Ah, okay, I take this for a week.

I'm good, it's done."

But modern medicine cannot exist without something like that.

You cannot go have surgery.

You cannot take your wisdom tooth without antibiotics.

We have modern medicine because of that.

So we have to work diligently in ways in which we can address this crisis.

And if you have drugs that are already FDA-approved that work and you know how they work, you have all of the safety on them already done.

So it can be adapted much faster than developing a new one.

- I, sorry, I have a thought based on sort of your talk too, of, like, the you know, the word antibiotics is anti.

And, like, you are killing the microbes with that.

But Vanessa, you know, her research showed that you don't have to kill something to stop its virulence.

And so that's another area, I think, that we can expand this sort of antibiotic but anti-virulence research into.

It's not just targeting killing the microbes, but targeting how they're actually carrying out that virulent activity, which is probably easier than eradicating a microbe from a population in the first place.

We have done some things looking at IBD-associated microbes and having increased levels of arginine in the diet can sort of both stop that microbe from activating an immune response associated with IBD, but also is helpful for that microbe to grow.

And so it's really interesting that you might actually, if you're helping some microbe's growth, it might actually prevent its anti, you know, pro-inflammatory response because maybe it's doing better, and so it doesn't have to activate those virulence pathways to increase its fitness.

So it's really sort of counterintuitive, but it makes sense in some ways of promoting growth to inhibit virulence.

So it's a fascinating, fascinating area.

- Vanessa: And there is also a very, a lot of very nice, beautiful data with MRSA showing that these types of strategies of disarming them without killing them actually work really, really well.

And there is actually even data on patients who got vancomycin-resistant MRSA infections and... Well, there's nothing anyone can do about that normally.

Right?

And in these types of cases, they ended up being treated with this experimental type of therapeutics, and they survived.

It worked, so the potential is there.

And, you know, I think in the field in general, we're all trying to look towards ways, creative ways in which we can expand armamentarium to combat diseases and also understand how to help with behaviors and addiction and neurodegenerative diseases.

Autism has a huge component that has to do with the microbiota, for example.

- Host: We have a online question from the audience online.

A member who's living with colitis and asking, "Is there any hope for me?"

[Vanessa laughing] - Vanessa: Well, there is hope for you.

[laughing] So, so with colitis, you can interfere with different diets, okay?

So you can basically try to, usually when you have colitis, you tend to have more E. coli than you should, which is kind of inflammatory in general.

So if you can change your diet for a plant-based diet, you can increase a member of the microbiota called bacteroidetes that breaks plant's carbohydrates like pectin and things of the sort.

And they become the majority.

And those are very good.

They actually prevent inflammation.

So there are dietary interventions that you can use.

I'm not advertising anything, but... That, you know, you can think about maybe Prozac or...

But this is not, this has to go through clinical trials because we are at the stage in which this is tested in animals.

Right, once it goes, once it jumps from a mouse to a human being, it's a total different ballgame.

- Attendee 5: Thank you.

[clearing throat] So I was wondering what kind of pathways and interactions between the microbiome and blood-brain barrier and those connections to your research regarding addictions and possibly depression and anxiety, like, what have you found regarding those biochemical pathways and connections and their associations with certain foods?

And are there any specific foods or maybe substances or supplements that you would personally recommend based upon your research?

- Vanessa: I'm cautious about recommending supplements and diets, okay?

Especially when you are in a very early stage still in animals.

In animals, I can say that at least for psychoactive addiction to cocaine, sarcosine seems to work okay in mice.

But from a mouse to a human, the jump there is the thing.

So their literature is beautiful.

And if you start going to old literature, in the '70s, there is this manuscript.

Okay, that was done in the University of Maryland by the Center for Vaccine Development, and they were developing vaccines against cholera, and they had clinical trials of the vaccines.

And at that time, they would have the vaccinated people, the unvaccinated people who were infected with cholera.

Okay?

And needless to say, several of the volunteers who would volunteer for that kind of study, a lot of them have come from jail.

And they had, like, their medical history.

And one of the things they noted was that heavy marijuana users were less susceptible to cholera.

There is, you know, it's one of the things that they noticed.

But...

But it was never followed up, right?

Because everybody was like, "Yeah, so what?"

You know, but there is, there was some sort of correlation, and it's a manuscript from the 1975, something like that.

It's old, but it seems like any diet that plant-based diets, I would say or Mediterranean diets are the ones who seem to, people seem to think that are the best ones.

- Margaret: But I would say too, it's still not really known how they're working.

- Vanessa: Yeah.

- Margaret: And so, you know.

You might see an average response to something over a population.

But what does that mean for you?

And thinking about sort of this personalized medicine approach.

You know, can we get more about understanding how exactly these things work, to more targetedly get at those interactions?

Yeah.

Oh, and I have got the signal that I think we are at the end of the time.

But I wanna thank you all so very much for your amazing discussion today.

It was lovely.

And thank you so much to Dr. Sperandio for an inspiring conversation.

And thank you again so much for joining us this evening.

[audience applauding]