- So we started with a very simple
hypothesis, which was that TLR4 signaling
is important for intestinal inflammation.
And so we reasoned that because
we were studying innate immunity,
we wanted to study an animal model
that really didn't necessarily require TNB cells,
that was dependent on bacteria,
and that was something early,
not like one of these chronic models
'cause we reasoned that innate immunity
maybe played a role in the early stages
of IBD or colitis in a mouse, and not the later stages.
So for that reason, we decided to use DSS,
which you know, at the time, we didn't
really know that much about animal models,
so we started with that.
And as you may know from other lectures that you've been to,
dextran sodium sulfate is, I don't know,
like a soap or something that they drink
and for some reason it disrupts the epithelial barrier
just of the colon, I would say,
for some reason, 'cause it goes down their entire
intestinal track, but it seems
only the colon to be affected.
It really does, wreaks havoc on the epithelial lining
and this results in colitis.
And it's dependent on bacteria,
it's ameliorated by antibiotics, et cetera.
So this is the model we chose to examine.
And so I just took a long time to explain to you
that TLR4 recognizes LPS,
there's this adapter molecule, MyD88,
that is important for lots of TLRs.
When we started these experiments,
and we used a knockout of these pathways,
the prediction might have been that
these animals would be protected against colitis, right?
They don't recognize bacteria,
so maybe they don't know there's a problem, okay?
The answer is both yes and no.
The animals are actually sicker
in the absence of TLR4 and MyD88.
This is one representation of sicker
that I can get away with showing to a GI audience.
The animals bleed significantly more
when they're knocked out for four or MyD88,
and you can see that based on their hemoglobins
that their mean hemoglobin drops significantly.
We did a lot of work to understand why that was.
So they have increased rectal bleeding, fine.
If you go a little too long with the DSS
you kill them more often than you would a wild-type mouse.
But in terms of inflammation, technically speaking,
actually they have less inflammation.
They're not able to recruit neutrophils to the intestine,
they're not able to recruit macrophages, as well
to the intestine, and as a result of this inability
to recruit these inflammatory cells,
we believe that's the reason why
they have bacterial translocation.
Bacteria escape the gut and are found
in mesenteric lymph nodes to a much higher extent
than they are in the wild-type mice.
In addition, they have a very clear defect
in epithelial proliferation,
so it's a defect that spans from the epithelial monolayer
of the gut all the way to the lamina propria
infiltrate that occurs in this DSS model.
So for simplicity, we can conceptualize
that the TLR4 knockout phenotype,
at least in the setting of DSS,
shows defects in leukocyte recruitment
and bacterial clearance, and that maybe is to be expected
if you don't have a receptor in the innate immune system,
but what was surprising was
this epithelial homeostasis and repair defect.
And I'll actually focus the majority of the talk on that.
So we hypothesized that proliferation apoptosis
in intestinal epithelial cells
was regulated by TLR4.
This is a proof of just the proliferation part,
you'll have to trust me on the apoptosis part,
and that's published.
This is an animal that's been sacrificed
after they've gotten a dose of BRDU,
which is a nucleotide that is incorporated
into their DNA if it's replicating,
and then you can easily come back
and use an antibody against it to tag it,
so a dark nucleus is the sign of a cell
that was proliferating.
You can see that before you do anything to a wild-type mouse
they're of course always proliferating cells
at the base of the crypt.
You give them seven days of DSS
and then you can see that there is an increase,
but after they've been through the injury of DSS,
in the recovery phase, you can see that there's
a very dramatic proliferative response
to try to repair that defect that's been inflicted.
But look at, by comparison, the TLR4 knockout
at, again, the comparable time point
at the same magnification.
I hope I don't have to convince you
they have very perturbed, very decreased proliferation.
At baseline, we don't really see,
if you don't injure the mouse,
the TLR4 knockout intestines look normal.
I'm not entirely confident that they don't have
some subtle abnormalities, but it's so subtle
that at least using level 1 techniques
we can't detect any abnormality.
So, you know, with this slide, I will summarize
a couple years worth of work.
To try to understand why it is
that these TLR4 knockout mice have defective proliferation.
And so what we found was that in the absence of TLR4,
when you injure the intestine,
you cannot express COX-2.
So no TLR4, you can't make COX-2.
And COX-2 is essential for
the production of Prostaglandin E2.
Prostaglandin E2 has many complicated
things it can do, among them it can activate
epidermal growth factor receptor signaling
in an indirect way by causing the release
of EGF-like factors, we happen to be looking
at Amphiregulin but there are
a couple others that it can do as well.
We're confident that this is part of the mechanism
because if we give TLR4 knockout mice back PGE2,
we can restore them back to a wild-type phenotype.
They proliferate and we can bypass the defect
by giving them exogenous PGE2.
Another way in which this might be working, as well,
is that maybe it's also the macrophages cooperating.
You know, the lamina propria's rich in macrophages.
These macrophages could also be TLR4-expressing,
respond to PAMPs, and they can then activate COX-2
and make PGE2 and still the epithelial cell
could benefit from this in a kind of a trans way,
not in cis but in trans.
Okay, so all of this led us to hypothesize,
well, if TLR4 is important for all of these processes
involved in expression of COX-2 EGFR and proliferation,
ergo this might also be involved in the extreme of that,
which is the development of cancer.
And so we hypothesize that TLR4 signaling
is important for colitis-associated cancer,
as distinct from sporadic cancer,
which is far more common in the population.
And so this is kind of a, you know, an example of that.
Unlike the idea that in sporadic colon cancer,
when Tim spends the rest of his day
in the endoscopy and he's looking for polyp,
sometimes raised, sometimes flat, that he'll remove
and for all intents and purposes that person is cured.
IBD must be different.
It looks different.
These areas that go on to become cancerous,
we believe are relatively flat,
probably not completely flat,
but they certainly don't become
broccoli sprouts and are easy to remove.
And in fact, that has been also demonstrated
in the genetic events that occur
with P53 and k-ras being much earlier in the cascade
in colitis-associated cancer and APC being a late event.
So we were interested whether or not TLR4, TLRs in general
are superimposed on this colitis-associated cancer pathway.
Are they involved in initiating events,
like reactive oxygen generation
and things that will mutate the DNA
and make it more likely that you'll develop
a monoclonal proliferating population of cells?
Or does it work later?
Once you already have dysplasia, maybe it allows,
helps in the growth of that already-present tumor.
The first thing we did was we asked the question,
is TLR overexpressed in colitis-associated cancers
and in ulcerative colitis, and so to answer this,
I think it's dark enough.
At this time of the morning with not enough coffee
I'd be reluctant to turn the lights down any further.
But for the most part, you can see on the left
that normal colon from,
this happens to be a completely normal person,
just has some TLR4-expressing cells
at the base of the crypts, and Scott Plevy has thought
that these are maybe entrochromaffin cells.
Whereas you can see next to it
is a colitis-associated cancer,
so cancer from two different UC patients,
the TLR4 expression seems to be much higher.
Obviously, I'm talking to a sophisticated audience,
and immunohistochemistry and immunofluorescence
can be problematic.
How do you really know
that this is what you're staining for?
And remember, this is archived tissue
so there are all sorts of things that go along
with dealing with archived tissue.
So at Mount Sinai we were lucky enough to have
frozen tissue from a colitis cancer
and the surrounding mucosa,
so this is a UC patient, the surrounding mucosa
is not cancerous, but it's also not completely normal.
You can see on the western blot,
and this is the right size for TLR4,
that the tumor expressed much more TLR4
than the surrounding tissue.
So at least, at the moment, I would say
we've probably done about 50 UC cancers
and it's about 80% of them have a high expression
of TLR4, whereas it's much, much lower in sporadic cancers.