NINR Advisory Council – 9/13/2016 -Effects of Chronic Stress on Microbiota-Gut-Brain Axis

[music playing]>>Wendy Henderson: So
wanted to just start with giving you sort of
a story of discovery, sort of where I’ve been over
the past, starting my tenth year in the
intermural program of NINR, where I’m very
grateful to be and grateful for your
attention today. So the focus of my lab is
the effects of chronic stress on the microbiota
and the gut-brain axis. So as we’re all walking
back into the room and thinking about what does
this have to do with me? So we can all imagine how
stress affects us and affects our lives, whether we’re
care giving, whether we’re receiving care,
or otherwise. And my focus specifically
is GI, gastroenterology, GI symptoms, hepatology,
and nutrition. And so I’m going to kind
of walk you through some of this but focus
specifically on this chronic stress effects
in the brain-gut axi.s As we’ve talking today
we’ve heard talks about precision and precision
medicine, something that I like to talk about as
precision science, especially from a
nursing perspective. And to remind you that,
you know, this concept is not new necessarily
to us in what we do. So let’s think about this. So consider your
prescription glasses. Consider blood
transfusions. From my GI, you know,
perspective, you know, we look at the biopsies and
we treat based upon what we see. Consider broader
applications raised by more recent advances and
imagine what was happened in the past five
to ten years. So when we think about
technology, big data, proteomics, genomics, this
is moving so fast, right? But what do we need? What do we need now, and
what is our role, and how can we contribute? And what is our niche? I’m going to tell you
about my niche and my team’s niche. We need development of
rigorous research programs to provide scientific
evidence needed to turn this concept into reality. We need recruitment of the
best and the brightest from diverse backgrounds
to join the team so that we’re not all alike and
asking the same questions but thinking differently. We need to develop
standards, standardized measures and resources for
generating and sharing data, but bridging that
gap between not just data given to patients, but
what do patients do with it? How do they share it with
their healthcare provider? How do we as clinicians
when they come to us, who do they talk to first? Nurses. What does it mean? So what is precision
science, which is hard for me to say right this
minute [laughs], at NINR mean? And, again, this is just,
you know, perspective, as we sort of move forward. But NINR has supported
scientists who are contributing findings that
can predict which people are most at risk for
adverse symptoms and conditions, monitor
treatment efficacy, guide interventions, improve
health and symptom outcomes. You’ll hear from all of us
in the intermural program in terms of tenure-track
investigators now, as to what symptoms we’re
interested in, what we specifically
can contribute. NINR, as Dr. Grady
indicated, has a model of symptom science in terms
of ways to guide research related to symptoms of
illness, injuries, and conditions. So we saw the symptom
science model, the complex symptom, the phenotypic
characterization, the biomarker discovery in
the clinical application. Remember this, embed this
in your thinking, and I’m going to put in, sort of,
my GI symptom perspective. So from a GI, or
gastrointestinal perspective, what are
those symptoms, they’re many and varied. They could be weight loss,
constipation, diarrhea, poor appetite, cachexia. Think of them. Stress induced. When you’re stressed, what
are those GI symptoms you might get? When you’re giving a talk,
what might you be having? A stomach ache? [laughter] And how do we phenotype
our patients? What is that phenotypic
characterization, so that we can say, “this is
indeed what you’re telling me, and how can
I quantify that?” And then that personalized
profile and on to the clinical application. So some key facts about
stress effects on intestinal health. So up to 20
percent of the U.S. population — 20 percent
— children, adults, the elderly — 20 percent
report stress induced GI symptoms. It’s the single most
common reason for emergency room visits and
is one of the top ten reasons for
outpatient visits. That’s a lot. 30 billion in costs
annually, many of which are unnecessary tests
and potentially risky interventions that
are not needed. And evidence shows that
chronic stress affects intestinal health, growth
and development across the lifespan. So let’s image a model
— okay, we’re talking science now so let’s
change our way of thinking here, moving on to the
afternoon session. I got to keep
you awake, right? But if you think about
mood, cognition, stress, and the different effects
that may have and how that affects the GI
tract, specifically. I’m biased, I think, you
know, the hindgut, you know, that’s the GI tract
when the brain rose out of the gut, so I kind
of focus on the gut. When Jessica and Leo talk
later they can have their perspectives. So, and where
can we intervene? So I’m very interested in
the GI tract, obviously, and the symptoms that are
GI symptoms that patients have. We’re also interested in
the brain-gut microbiota access. So what am I doing? What have I been doing in
the intermural program? So, I have a natural
history protocol that follows patients over time
that have GI symptoms with no organic pathology. So they’re histologically
negative, they’re normal weight and overweight, but
they have chronic symptoms for greater than six
months of abdominal pain or GI symptom distress
for which they have no explanation. They’re males and females,
we control for ovulatory effects, ages 14 to 35 in
this particular protocol. And they continue to have
these GI symptoms that are stress induced. So what was the first
thing that I found? They have these symptoms,
but we didn’t have a good way to quantitate them. So what we needed to do
was develop a way where patients could tell me or
my staff how they were feeling, where they were
feeling it, and be able to capture these symptoms. So this innovation
is called the gastrointestinal
pain pointer. We developed this
technology in house at NIH; we beta tested it and
validated it, and recently published it. The technology is
commercially available but is for free for use. It’s paid for by the
taxpayers for the people and is available
through NIH. So what this does is
basically patients can pick what they look like,
if they’re normal weight, if they’re overweight —
for pediatric patients, we have tanner staging as
well — and it captures in real time, they circle
where it hurts, they dial up how much it hurts, they
use word descriptors, and then a pop up field gives
heart rate and blood pressure. And then it’s got a
reporting mechanism and an automatic reminder so that
in-house when we do a stress test where we
induce their symptoms, we can capture when their
symptoms occur overtime as well as the other
effects there. So the next thing we
needed to do after we created a way to better
quantify their symptoms, both objectively and
subjectively, was to experimentally induce
their symptoms. And that’s something that
can be done, not easily anywhere else except in
the intermural program of NIH. So I’m very grateful to
have this opportunity to do this, and what we
did is we developed a brain-gut test solution. We got to name it, too,
so that was kind of fun. But basically it’s a
combination of sugars. It’s a 100ml’s the
patients drink, and it measures intestinal
permeability. Lactulose, mannitol,
sucrose, and sucralose are in the compound. Patients drink it, they
get GI symptoms, and then we measure their symptoms
at 11 time points over a five-hour period in house
at NIH while they’re hooked up to a
halter monitor. And then we calculate
the excretion, or the metabolism, of these
particular sugars via urinary secretion
via mass spec. This method, which we came
up with, we have published and is used here at
the clinical center. And the same method
is also used in other hospitals where we make up
the test solution and then we send it out and
teach them our methods. We found significant
differences in our particular cohort of patients. Counterintuitive, but we
actually found a decreased urinary excretion of
sucralose, which is a non-absorbable sugar that
permeates the colon. So this was very
interesting, especially in our irritable bowel
cohort, which is IBS compared to control. And this is published
if you’d like more information on this. So moving on. So we’ve got personalized
profiles, and what became very interesting to us was
that we could predict from the oral microbiome of
patients how they would respond to the test
solution and whether or not they’d get pain. And so it became very
interesting that patients with certain profiles were
actually 20 times more likely to get symptoms
when given different sugars. So the next piece, the IVP
is the induced visceral pain, and this is
lactulose which is another one of those
non-absorbable sugars that’s in the
test solution. Lactulose is interesting
to me — and you wonder why, “why you’d you
pick lactulose?” Right? “What do you
use that for?” And, of course, you in the
audience know what we use this for, for treating
constipation as well as hepatic
encephalopathy, right? Brain-gut. Lactulose isn’t broken
down by the human body; it’s broken down by the
microbiome in the colon. So, creating an algorithm
that actually shows that you can predict the
induced visceral pain but more so particular bugs,
or microbiota, that are associated with patients
who have increased permeability and also
get pain from the test solution. And this is just another
association in terms of the different microbial
taxa, some of which are pathogenic that are
associated with GI symptoms in our cohort. So what does this mean? And we don’t know fully
what it means yet, we have a lot of work to do. But what we’re very
interested in is the host-microbiome
interaction. And what, specifically in
our lab with Dr. Nicholas Forrey [phonetic sp],
who’s here in the audience — raise your hand, Nick. Hello. I’m sure many of you went
to his poster as well — is what is the function
of these microbes? Not just what is the
particular microbe, but what are they doing, and
how as a group, how are they affecting humans
and intestinal health. And so what we found
was that butyric acid producing microbiota
actually are predictive not only in the peripheral
gene expression from our patients, but also in
the microbiota that were associated. So we also find a
significant weight effect. So patients who had
GI symptoms that are phenotypes specifically
with irritable bowel syndrome actually had an
increased — well, it’s not really a difference in
terms of their diversity and relative abundance,
but a differential expression in terms of
what they’re doing, and I won’t go into all the
details of that as we recently published
this in gut microbes. So the next thought was,
“Okay, Wendy and Wendy’s team, so you found this,
but how’re you going to validate this, and what is
the functional evidence that this is real?” And so what we’ve done is,
together with John Wiley’s [phonetic sp] group from
University of Michigan, looked a chronic water
avoidance stress model in animals where the water
comes up, you have animals on an island for one hour
a day for ten days, and they get stressed. I wouldn’t be stressed if
you put me on an island for one hour a day
[laugh], I’d be kind of happy, but — and then we
look at — after they’ve been chronically stressed,
we look at the microbiota, the whole gene expression,
as well as ion sequencing for the genes, associated
in the colonic area. So what did we find? So what we found is
similar butyric acid producing in terms of the
functional relevance, microbiota are associated
with the stressed animals compared to the
control animals. So that’s very
interesting to us. So we want to know, you
know, what is this, and why is there an affect in
the epithelial cells of these animals as well as
the oral microbiome from our human models. So yet again, more
validation, more proof. So we moved on to the
effects of the epithelial cell barrier function. So what we did is we grew
up epithelial colonic cells in the lab, we
chronically stress them with dexamethasone,
and then we added the byproduct of the
microbiome that we found from both the animal and
human models, arbutirate. And then what we looked
at is trans epithelial electrical resistance,
which is kind of like intestinal permeability,
but really, it’s using these two little chopstick
kind of things with electrodes on them to
look at how ions permeate between the cell layers. And what we found is it’s
not just the microbes alone or the stress alone,
but the interaction of the stress and the microbiome
together that caused this change in permeability. I’m kind of simplifying
this in such a way and I hope it’s understandable. So the next thing we
needed to do was, we found this difference in the
electrical ion flux or the permeability of the cells,
when you chronically stress them and put the
byproduct of the bacteria. But we needed to
have more evidence. So here’s some
more evidence. So what we did is we
stained the tight junctions and the tight
junctions are what — when they’re released, these
tight junctions, that’s when the ions’ flux flows. And so when we stained
them for Z01, which is the red zonula occluden, which
is a scaffolding protein that links together this
— it’s a trans membranous tight junction protein you
can see here, and we also stained these cells for
occluden, here in green. And what these are, this
is untreated versus chronically stressed, and
then chronically stressed with dexamethasone, and
let me show you a little bit better. And what you see — and
this is the Z stacking video — so that untreated
is what’s moving right now in terms of the video. And you can see the tight
junctions, which is the yellow, that’s sort of the
merge overlap, and you see the nice blue staining of
the nuclei, so really this is nice healthy cells in
terms of tight junctions from the colon. But when you chronically
stress the cells — so now I’m turning on the other
slide here — you can see the tight junctions,
you’re sort of going through the Z stack
looking through the layers of the cells. And then you see these
sort of nice vacuoles coming here form the
stress affects, and then you see these tight
junctions starting to fall apart. So this was — and we can
bring the lights back up a bit now — so this was
very interesting to us, and, of course, I’m very
interested in what are those vacuoles and what’s
in there and what is that? And what can we do with
that and is that something that we need to further
investigate for sure. So the other thing was, we
went back then, okay, to our human samples. We thought, okay, can we
look for other molecules that are just as small as
those vacuoles and those tiny purses that are
carried around that cross the blood brain barrier
in the GI tract? And we actually isolated
from our human samples, the same patients who had
the oral microbiome and so forth work done, and we
looked specifically in the irritable bowel patient’s
cohort who had evidence of microbial translocation,
which means that there’s flux sending a toxin. And we stained the
extracellular vesicles; we isolated them and then
stained them, specifically looking for these markers. So this is very exciting
work, and Sarah Abby [phonetic sp] who’s in the
audience here — Sarah, if you could put your hand up
— learned how to run an electron microscope and
shared facilities with Heart and Lung Institute,
and we stained for those but not only that. We found evidence of muc
1 and muc 2 in the plasma circulating from the
patients and these are markers of the
epithelium of the gut. So this is very exciting,
this is still a work in progress,
unpublished data. But we have evidence that
individuals who have stress induced GI symptoms
and get pain from our test solution actually have
circulating markers of immune markers that we can
show that are associated with their symptoms. This is sort of very
exciting information. So we’re moving through
the symptom model, and I’m finishing up my talk, and
we’ve been able to, you know, invent new ways for
patients to tell us how they feel, even if they
can’t talk or if they’re intubated, they can still
say where it hurts and how bad it hurts, and
we can use that. We have a way to look at
microbial translocation and intestinal
permeability. We also are looking
at the microbiome. And then furthermore,
in terms of clinical applications, I got
permission to share with you from our creative
partner one of our newest inventions. So, some of you may not
have been here — I think many of you are kind of
new — but three years ago when I came here, I said
my biggest dream in intermural program was to
be able to see a patient that wasn’t doing well and
had significant symptoms, and within five minutes be
able to tell them what I found that was wrong and
have a way to treat them and not lose them to
follow-up, something that haunts me as a clinician. And so what we have is a
paper based diagnostic, and this was recently,
last year, a non-provisional worldwide
patent was issued for this. And the goal, for under
a dollar, in under five minutes, you can
help a patient. So, coming back. This is our
research in action. This is our story
of discovery. Other on-going studies,
just for examples, one of our Harvard collaborators,
we send out the GI pain pointer, we send out the
test solution, we send out our methods, and they use
this at Harvard in HIV protocol in a randomized
controlled trial. Other on-going analyses,
more high sensitivity mass spec for sugar
quantification, and future studies, just how to
target stress induced GI symptoms and improve
outcomes across the lifespan. So I have a lot of people
to thank, a lot of people in the audience, and
thank you very much. [music playing] [applause]

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