My name is Lalita Ramakrishnan.
I'm a professor of Immunology and Infectious diseases at the University of Cambridge.
And, in this lecture, I'm going to continue to tell you a little bit more about tuberculosis,
focusing on a structure called the tubercle.
Now, just to recapitulate the lifestyle or the life cycle of Mycobacterium tuberculosis,
which is the causative agent of tuberculosis, the bacterium that causes it, the bacteria
are exhaled by... in coughs by people who are infected, get inhaled by individuals near
them, and then they enter cells that are called macrophages.
But what happens next is that they... they induce these macrophages to form structures
called granulomas, and these granulomas can become quite elaborate.
At first, they're comprised just of macrophages, but then many other immune cells come in and
they can form of a fairly complex structure.
And another thing to note is that the macrophages within this granuloma undergo a specialized
differentiation called epithelioid transformation, where they form these finger-like, interdigitated
projections between each other to form a very compact, organized structure.
Now, this structure... pathologists call these structures granulomas, and granulomas were...
are... are associated with many, many, many diseases, both infectious and non-infectious.
And they can often be quite pathological.
But granulomas... but... but the single biggest cause of granulomas is tuberculosis.
And, in fact, granulomas were first discovered in the context of tuberculosis in 1679, some
200 years before the bacterium Mycobacterium tuberculosis was... was discovered.
And, in fact, the microbe... the... the structure used to be called a tubercle, and obviously
that... both the bacterium and the disease are named for the granuloma.
So... but it turns out that granulomas are very primitive structures; they've probably
evolved... there... they're present even in lower vertebrates in a... in a more primitive
form, and they probably evolved to wall off foreign bodies -- you could imagine a thorn
being... being circumscribed by that... by such a structure until it's...
it's sort of dissolved.
But these so-called foreign body... body granulomas have a very low turnover of macrophages; the
macrophages just come and sit there and it's not a particularly inflammatory structure,
or so it's thought.
In contrast, the types of granulomas that form with tuberculosis, and pretty much any
medically significant granuloma, tend to be high turnover granulomas where there's a rapid
death of macrophages and a repopulation by new ones,
and they can be very inflammatory structures.
There are many traditional animal models that are used to study TB.
The oldest ones are the ones... the rabbit and the guinea pig, which were used by
Villemin and Koch, respectively, at the time that they discovered TB.
They used these animals to... to pass the bacterium from one animal to another to show
that it was associated with... with TB.
The most commonly used model now is the... is the mouse.
And this makes a lot of sense because mice have a wonderful array of
immunological and genetic tools.
One issue is that mice don't... that most mice... mouse strains don't develop the...
the nice, tight granulomas that are associated with human disease, but there are some recently
identified mouse strains that do, and so those could actually be quite good.
Another more recently used model is the non-human primate, and this is a good model because
it really recapitulates human disease.
But the problem is, of course, they're expensive, there are ethical considerations, and obviously
cannot be used widely.
So, in... on this backdrop, my story and my engagement with TB and the granuloma came
when, as a postdoctoral fellow at UCSF -- I was a clinical infectious disease fellow and
had to do a postdoctoral fellowship -- I approached Stanley Falkow at Stanford to... to go to
his lab and study TB, which he didn't study at the time,
but he studied many other bacterial pathogens.
And Stanley said to me, forget it, I don't have the specialized containment facilities
you need to study TB, which is a human aerosol pathogen and, besides, he said, TB grows so
slowly I'll be dead before you get your first result.
And that was in 1991, and I'm happy to say he's still alive, and... and...
and quite well.
And... so... what he told me... he gave me an insight and he said, look, there are other
strains of mycobacteria, there are other species of mycobacteria that are pathogenic in other
animals, that are natural pathogens of other animals.
And he said, I...
I'm pretty sure there are these ones of... of marine life of fish, because I've seen
people get them who were fishermen.
He had worked at Brown and knew that Portuguese fishermen got these... this disease... mycobacterial
disease on their digits and on their soft tissues.
And so I went off to the UCSF library and looked at this very classic manual.
It's called Bergey's Manual of Systemic Bacteriology, and I... and I found what he was talking about.
He was talking about... he was probably talking about a bacterium called Mycobacterium marinum
that was thought to be a close relative of human TB, of the human TB bacterium, and it
gave fish TB.
It turns out that it also infects humans, and this has been known since the 50s, and
I can personally attest to it.
And it gives humans disease on their extremities, as Stanley already knew, and... and many clinicians,
particularly dermatologists and infectious disease clinicians, already know this.
But if you look inside that lesion, you'll see a classic granuloma and actually in many
cases it can be indistinguishable from the granulomas caused by the human TB bacterium.
But, of course, we know that this bacterium also infects fish, and it was first identified
to do so in the Philadelphia Aquarium, where in 1926 fish were dying of some mysterious
wasting disease, very similar to human TB.
And when they tried to culture these fish to see what bacterium they had, why were they
dying?, they couldn't culture anything, but when they looked at the fish by histology
they could see these classic red snapper bacteria that looked very much like TB.
And then Aronson had the bright idea to culture the... to try to do the cultures at a low
temperature that was commensurate with... with the low body temperature of the fish,
and then he was able to cultivate, he was able to culture Mycobacterium marinum.
And, since then, we've had Mycobacterium marinum sequenced at the Sanger Center, and it turns
out to be the closest genetic relative of the human TB bacterium, so I guess we also
got quite lucky.
And it turns out that Mycobacterium marinum also infects zebrafish.
Zebrafish are a pet develop... a pet organism of developmental biologists and are a natural
host to Mycobacterium marinum.
So, I've got for you, here, down below in... in the bottom panel, the... a human TB granuloma
stained by hematoxylin and eosin, which will only stain the host cells but not the bacteria,
and what you can see is that you've got a nice organized structure, which is cellular,
as evidenced by blue nuclei on the edges, but in the center where that arrowhead is,
you'll see that the structures become acellular, because it's undergone necrosis, just as we
know that human TB granulomas do.
But here's, now... let's take a look, now, at the... at a zebrafish granuloma, and in
this granuloma you can see... which... which I've stained, here, with a stain that also
stains the bacteria, you can see that it looks very similar and it's a nice cellular organized
structure and you can see that there are a few bacteria within macrophages, but where
the macrophages have necrosed there are tons of bacteria,
which is exactly what you would expect.
But the great feature of the zebrafish that makes them so enticing to developmental biologists
is that they have a prolonged larval phase when they're transparent.
And so you can actually watch things happen and people watch developmental processes happen,
but... so we asked, well, can we put in bacteria and watch infection happen?
And so, they have a cavity that's called the hindbrain ventricle, which is the equivalent
of something in our brain, close to our brain called the fourth ventricle, and so we put
in some bacteria there -- I've shown it to you with an arrowhead, there -- and what we
saw very quickly was that macrophages came, and you'll see the macrophages sort of chasing
after the bacterium like a cat after a mouse, and eventually you'll see this mac... mac...
macrophage gets it.
And there you've got an infected macrophage.
You can now follow these infected macrophages out of the cavity -- this is a few days later
-- and you can see that it's just moseying along.
The bacteria have grown in the macrophage and -- because it's a permissive macrophage
for the... for the... for the bacterium -- and there it is.
But what was really exciting to us was that you could see, within a few days,
a granuloma form.
And here you can see that we've got a granuloma that's already formed and what you're going
to see, where that white... white arrow is, a new uninfected macrophage is going to come
and then it's going to enter the structure.
So, watch this.
See?
There it comes, and it's going to squeeze its way in between and get in there.
So, the granuloma is a highly chemotactic structure
that is recruiting new macrophages to come to it.
And then we could show all this by... by engineering fish that were transgenic, so that they had
green florescent macrophages and red fluorescent neutrophils, which is another cell type that
is somewhat involved in granulomas, but not as much as macrophages.
And what you can... and now we've infected the fish with blue fluorescent bacteria and
you can see that the... that we've got a nice tight bona fide epithelioid granuloma with
infected macrophages.
So, this was good.
As we were developing this model, my colleague and friend, David Sherman,
made a suggestion to us.
He... so, it's... people have been searching for virulence determinants in mycobacterium,
and one exciting discovery was that a specialized secretion system called the ESX-1 or RD1 locus,
which I've shown in white in that top panel, the white genes in the top panel,
were involved in virulence.
And this was very exciting because these... it turns out that this was the locus that
was missing in the attenuated vaccine strain, BCG, that was made by serial passage in...
in the 1920s, and now we finally knew the molecular basis of its attenuation.
So, Mycobacterium marinum, not surprisingly, has a locus that looks virtually identical.
And David kept telling me, look, make a mutation in this and let's see how it really works,
because everyone knows it's attenuated, but a lot of these animal models are black boxes
because you only get to see the end result, and he could see that we would be able to
get some insights about the actual sequence of what was got... what was different.
And, when I was a bit slow to do this, he actually had someone in his lab make the mutation...
the mutant for me, and he gave it to us and he said, take this.
So, at this point, we were sort of shamed into doing this quickly, and we was
Hannah Volkman, who had joined my lab at... as a graduate student, and Hannah showed very quickly
that, yes, if you put this mutant into zebrafish larva, it was attenuated.
The animals didn't die and if you looked at the bacterial counts you could see that the
bacteria didn't grow as well.
So, this was good because it showed us that it was behaving just like you would expect.
But, here came the surprise.
And, at this point... by this point, Hannah had recruited some of her colleagues -- Dana
Beery, on the left, who was a technician in the lab, and Hilary Clay, a graduate student
who was Hannah's very good friend -- and she got them to join in this... in this quest
to see what was going on.
And what they found was something quite interesting.
Because, if you look at the fish on top, they are infected with wild-type bacterium, and,
if you look at the close-up on the top right,
you can see that a nice big granuloma has formed.
But if you look at the mutant, what you can see is that, even if you inject many, many
more bacteria, just to compensate, so that you get more... as many bacteria as with the
wild-type, the macrophages pack up with the bacteria, as you see on that bottom-right
panel, but there's... they don't form granulomas.
Now, this seemed opposite of what you'd expect, because if... if granulomas are good for the
host, as what everyone in TB... in the field of TB thought... people have thought that
the granuloma is a critical host protective structure that walls off the bacteria and,
while it's not always successful in eradicating the bacteria, it sure as heck tries to do
so, and is... is... is pretty good at it.
Now, if that's the case, then we should see more granuloma formation with that mutant,
but we saw less.
So, Hannah took a close look at this and she was able to observe fish as the granuloma
formed by serial imaging, and what she showed was that, when the granuloma formed, the number
of infected macrophages went up dramatically, as did the number of bacteria.
So, the granuloma was actually promoting growth rather than restricting growth.
So, why might this be?
Because here we are saying that a... the... you know, this... this immunological structure
that really should be killing the bacteria is actually promoting growth.
And the answer to this came of... both from Hannah's work and from a new graduate student
who joined, an MD/PhD student, Muse Davis, and what we found was happening was that,
when there's an infected macrophage, when new macrophages come to it, for some reason,
if they have that ESX1 locus, the bacteria are spreading quickly from macrophage to macrophage.
You can see, within 48 hours, you've gone from one infected macrophage in that top-left
panel to many infected macrophages, whereas if you didn't have that locus, if the bacterium
didn't have that locus, then that one mac... macrophage just remains one great big macrophage.
And the bacteria are just growing in it, but obviously they're not doing as well as if
they can spread to new macrophages.
And so it turned out that the bacterium is
using this locus to spread from one macrophage to another.
Now, how does this happen?
What... what Muse found was that, if the initial macrophage was infected with bacteria that
contained this locus, then somehow that macrophage was able to exert a rapidly chemotactic effect
on... on macrophages around it, or... or even far away, so that they came.
And you can see that the macrophages in the top panel have these protrusions that reflect
that they're highly chemotactic and are responding to a chemotactic gradient, and our racing
into this structure.
In contrast, if that initial macrophage didn't have this locus, then the incoming macrophages
are coming in very, very slowly, and they clearly are not experiencing a chemotactic
gradient; they don't have that big... great big protrusion.
And, even once they get into the granuloma, they behave very differently.
The wild-type macrophages move far and wide within the granuloma, and rapidly, whereas
the mutant macrophages, the few that do come, are just sort of sitting there
like bumps on a log.
And this is the kind of movie that gave us that insight that I just talked to you about,
that macrophages are used... exploited by the bacteria to spread from cell to cell.
So, what happens is that, when a given infected macrophage packs up with... packs up with
bacteria, because the bacteria can grow within it, it undergoes an apoptotic death, where
it's dead but it's preserved its membranes.
And now what happens is this dead cell is recognized by the incoming macrophages, that
come and eat it.
And I'm going to show you an example, here, of where one... one dead macrophage with the
white arrow is going to be engulfed by an incoming macrophage.
So, watch this happen.
It's... it... you're going to watch it, it's going to come from below, it's kind of like
that movie Jaws, where the... you know, the shark comes from below.
And, look at it, it's eating it bite by bite.
And this is why macrophages might be called what they are -- macrophage for "big eater".
And now you've got... this macrophage has been eaten by a new macrophage.
But you're going to say, wait a minute, why would this spread the infection?
You've gone from one macrophage to another macrophage, so all you've done is conserved
the bacteria.
But it turns out, when Muse looked closely, that, on average, a given macrophage, given
infected, dead macrophage, was eaten, on average, by 2.3 macrophages every 24 hours.
And so you can imagine... you can see how the bug is using the macrophage to expand
its numbers.
And not only that, but we showed that the bacterium also induces the death of the macrophages.
Here's a TUNEL stain on the left and this... this death... there are probably many bacterial
determinants that do this, but one of them is that self... same locus, ESX1, that also
induces the macrophages to come.
So, it's got a two-pronged effect.
It's inducing death of the infected macrophage and, separately, it's recruiting new macrophages
to come to it so that they can engulf the dead macrophage, and... and produce infection.
So, to summarize this, let's take a look at what happens in the mutant first.
Because, in the mutant, the granuloma might actually be functioning as a host-protective
structure, as it might be meant to be.
It's you've... you've got an infected macrophage, it dies at a slow rate, macro... new macrophages
are slowly recruited at a... at a respectable pace.
And, now, they can eat the new macrophage one by one on one, and there's some time for
the cells to also kill the dying cell, to also kill the bacteria before they're eaten,
so you could imagine there's an attrition of bacterial infection.
This might be why the BCG vaccine strain is attenuated.
But, paradoxically, instead of sort of thwarting these host-protective processes, as you might
imagine a pathogenic bacterium might do, the... the pathogen actually accelerates these processes,
so it converts them from being a host-protective to a host-detrimental process.
So, simply by speeding up the rate of cell death, and speeding up the recruitment of
new macrophages, it's... it's just using the macrophage niche to spread in from cell to
cell, and therefore expand itself in the granuloma.
And this is quite a nifty thing to do, I think.
So... okay.
But the question now is, how do... how do... how does this ESX1 locus induce the recruitment
of new macrophages?
And so, for this part, Hannah was joined by Tamara Pozos, who was a pediatric infectious
diseases fellow who joined the lab, and together they did a microarray where they looked at
fish that were infected with wild-type or mutant bacteria to identify host genes that
might be different between the two.
And the gene that stuck out was matrix metalloproteinase 9.
And this is an extracellular... an... an enzyme of that... that models...
remodels the extracellular matrix.
And they were even able to show, not only by transcriptional analyses but also by doing
a gelatinase assay on the fish for the actual activity of this enzyme, that MMP9 was induced
upon wild-type infection but not upon mutant infection.
Now, that's fine.
But if MMP9 is... induction of MMP9 is responsible for the ESX-mediated acceleration of macrophage
recruitment, then, if you make a MMP9 mutant, that mutant should be attenuated even with
wild-type infection.
And, sure enough, when they looked they saw that the MMP9 mutant, which is shown on the
bottom, there, that fish, was attenuated for infection, and it had very few granulomas.
So, it was behaving just like the bacterial mutant and therefore it was the partner.
So, so this was nice and then... but then we started to look into what MMP9 does and
then it turns out that MMP9 is involved in the pathogenesis of arthritis, and cancers,
and other inflammatory conditions, and often in those cases the mac... it's the mac...
it's a macrophage that is the bad actor, that is making MMP9 and... and causing trouble
in these lesions.
So, of course, we thought, well, okay a macrophage gets infected with the bacteria it now induces
MMP9 in the macrophage, and now that... that is secreted and calls in new macrophages.
But when we did in situ analyses there... to look at where the MMP9 was being made,
here is a granuloma, and the MMP9 is labeled green and the macrophage... macrophages are
labeled red, and what you can see is that the MMP9 is not in the... in the macrophages
of the granuloma.
But, rather, it's in the epithelial cells surrounding the granuloma.
And so what... what seems to be happening is that an infected macrophage secretes something
from that secretion system that goes and talks to the epithelial cell
and induces it to make MMP9.
And the MMP9 now calls in new macrophages, and this... this... this strategy was called
"subversion from the sidelines" by my friend and colleague, Bill Bishai at Hopkins, and
I rather like how he put it.
So, why might that be?
You could imagine that the bacterium wants to tamp down immunity in the infect... in
the actual... in the macrophage itself, because it has to survive in there.
So, it's doing that and, meanwhile, it's inducing an inflammatory program in a neighboring cell,
so that it can bring more macrophages, infect, and then subvert them.
Okay.
So, but... but... so this is so... so this is how the bacterium uses the innate immune
phase of the granuloma to promote its... its growth and expansion.
Of course, then the bac... the granuloma matures and other things happen and, as I told you,
one of the things that happens is epithelioid transformation, these tight interdigitated
projections, that too has been known for, oh, a hundred years or so, and that very reasonably
was thought to be a host-protective mechanism that would sort of wall off the bacteria and...
and perhaps be... somehow help the host, despite all these strategies of the bacterium.
Well, very recently, work from David Tobin's lab, also done in the zebrafish, that this
too turns out not to be the case.
Mark Cronin and David Tobin have shown that epithelioid transformation of the macrophage
is also something that the bacterium is benefiting from.
If they inhibit it, then they can... they get less infection than if it's there.
And, of course, they're working out the details of how this might be, but what it's telling
you is that practically every step that we might predict will help the host can be taken
advantage of by the bacterium.
So, in my first lecture, I told you that most people actually clear infection and they do
so after the adaptive phase of the... of the granuloma has kicked in.
So, it's very clear that... that at some point the granuloma can fight back and can... can
eradicate or at least suppress infection.
And many, many people who specialize in the areas of adaptive immunity and TB are... are
working on this.
But I want to close by saying that, while the adaptive immunologists may not know as
much about this as they want to, and they're working hard to figure it out, the bacterium
seems to know quite a little... quite a bit about this... these immune mechanisms, because
what these people so... who worked on it so far can tell you is that it tries really hard
to delay and inhibit adaptive immunity, so that the adaptive immune elements that come
into the granuloma do so late.
And this gives time for the mechanisms that I've just been telling you about to help bacteria
expand in the innate context.
I'll close by thanking the many people whose research I've described to you -- they're
both students and from my lab, as well as colleagues and collaborators from outside
my lab, and, indeed, from across the world.
Thank you.
