Animal Models of Air Pollutant-Induced Non-Atopic Asthma and Rhinitis

By Adem Lewis / in , , , , , , , , /

– What I’m gonna talk about is how we use
the large-animal model here the non-human primate and rodents to look at specific disease,
this disease is asthma and rhinitis, in the context of how they’re affected by air pollution. So we’ve been fortunate over the years to
work with many extremely talented collaborators. And we’ve had funding for over 30 years from
various funding agencies, from the EPA to NIH, and have used, also, just recently, we
have had a large seven-year grant that was looking at the health effects of air pollution
using laboratory animals but also human subjects. I’m gonna focus today on this problem of what
is the best model, animal model? First of all, that maybe is not a very good
term. So the term model, maybe we should be thinking
about experimental models and disease models like Charlie talked about and then look at
the appropriate model to use rather than just starting with a mouse or primate or another
mammal. And so the reason for that is as a pathologist
and anatomist, I wanted to really target in what is the best anatomy in subjects that
I want to make progress in in understanding the disease prevention or treatment. And so these challenge questions that you
were given were really good ones, I think. And we’ve already heard that Dr. Hyde really
did an excellent presentation about the advantages of the large animal models over the rodents. I’m gonna bring that in too, but also how
we can use rodents to also provide us information. And we can go back and forth, but really concentrating
on the dates. And then what are some of the changes, working
with these or challenges, working with these models that we have to overcome. And as I’m sure a lot of you already know
that. And I’m gonna kind of talk about all of these
throughout my talk, also show how important infrastructure is here. And I couldn’t have done any of this work
if I didn’t have the proper infrastructure to conduct it, especially with non-human primates. And then also, the final thing is how do you
see these models increase in coming years, and I’ll try to bring us back. The history of my journey, starting out as
a veterinarian, a veterinary student, all the way through where I am now. So I’m not gonna take 30 years to get this,
but I’m just gonna put this down really quickly. But what I’m gonna talk about, first of all
identify the problem. Look at comparative airway responses to air
pollutants, we’re gonna use just one, ozone. And then I’m gonna talk, tell you about some
new paradigms that we have learned over the years, especially very recently, about certain
cells that may control it. And then the future, for using either non-human
primates or maybe if there’s another model, that we’ll talk about. So this diagram kind of shows you how we started. Actually I started with a large-animal model,
the non-human primate, and identifying effects caused by air pollution on the airways of
these animals, then carried the research into laboratory animals. And eventually, and I’ll show you how, I think
we need to move with that into the large animal. But ultimately, it’s not about us and here. It’s about, as was emphasized in the previous
talk too, it’s about the condition, or the person that we’re trying to help through our
research. And I think that always adds a bonus. And I’ll show you, this translation, as Dr.
Hyde pointed out, this is the major gap. We haven’t done a very good job of that. We create lots of data, but we don’t translate
it to the human condition or to the laboratory animals’ diseases. And there’s a tremendous gap here, that’s
been identified. So what’s the problem, that I been studying. That’s the problem, the air pollution. And this continues to be a major problem. Almost half the people in the United States
still live in counties where there’s unhealthy levels of ozone, particulate matter. You can see, this is LA, but the health burden
is tremendous. And this just illustrates the percentage of
mortality that happens in different areas of the country. You can see in the mid-west, the south-east,
and the east coast, the west coast, that’s where the primary health problems are. And if we look at, similarly, with compliance
for just one pollutant, like ozone, this kind of matches up. And so you can see that there’s a huge problem
with air pollution still in the Los Angeles area and out in the east, and even here, in
Michigan, in certain areas and counties. But the even more dramatic problem is in China
and India, where this has been, like 4,000 people died per year, per day, something related
to pollution. So this problem is actually significant in
India now, actually it’s about three times the problem that it’s been. So I’m gonna take just an example now of commonly
used laboratory animals to understand the disease process and show you how it’s related
to traffic diseases and asthma. So we’ll talk about ozone and its health effects. This is a toxicant gas, it’s extremely, it’s
found in photochemical smog. It’s created as a secondary pollutant. And as I pointed out, many people live in
areas where the concentrations are higher than they should, and these causes respiratory,
pulmonary function deficits, airway inflammation, and remodeling. It also causes cardiovascular diseases. Now it’s been identified, along with particulate
matter, in reproductive diseases, neurological diseases as well. So it’s sort of the new cigarette smoke, where
you have all these systemic problems due to this pollutant, and air pollution in general. I’m gonna talk a lot about asthma in the next
few minutes, and rhinitis, and I know you can’t see it very well, you can see this is
kind of a mechanistic framework of how this air pollution affects asthma. Start down here. Here’s asthma, and we’ve known for years that
air pollutants can exacerbate the condition. And so that’s not really, that’s certainly,
we have to reduce air pollution’s exacerbation. But we also understand now that pollution
can also affect the onset of asthma, or even the development of asthma. And I’m gonna talk about what we have learned
from these animal models about this aspect. And actually, John Balmes, a friend of mine,
wrote in Lancet, he emphasizes pollution in certain areas. But I’m gonna show that there’s a total different
phenotype, in terms of inflammation, immunology, that we weren’t, that we have to understand
better. And we’ll show here, but I think we can do
this through large animal models as well. So the focus is next gonna be, this talk’s
gonna be on asthma, the most common chronic respiratory disease of children. Six million children have asthma in the United
States. 16, 17 million asthmatic adults, and 300 million
worldwide. It’s not just one disease. So it is actually now described as many phenotypes. And the emphasis of this research now is really
to target what type of phenotype the individual person has. So asthma is looked at as a reversible airway
obstruction, in terms of the wheezing pathology shows excess mucus and airway remodeling. There is actually an increase in the number
of cells and also fibrosis around the airway. But it’s a reversible condition. But chronically. So it comes and goes. So there are some phenotypes that have certain
cytokines, like what we call type-two cytokines. They’re very high in their expression. That leads to a number of pathologies in the
airways I’ll talk about. But there’s other phenotypes that are very
low type two. And it’s not just allergic asthma, but also
non-allergic. I was telling your seminar speaker last week,
from the UK, that children, at least 40% are non-allergic asthma. So there’s early onset, late onset, there’s
different inflammatory cells. And these are now all identified as different
phenotypes that we have to characterize. The emphasis of this paper and several others
is that we have to have better animal models to look at this because you can’t do it in
the human subject. So this is actually material I got from the
National Primate Research Center site, and it also emphasizes this emphasis, in the United
States and elsewhere, on laboratory animals. Very few studies in non-human primates or
other species of mammals. The monkey is a good model. Mainly it will emphasize because of its genetics,
we share over 90% of our genes with non-human primates. And there are certain areas, one that was
organ transplant, as well as looking at heart disease, brain disease, cancer, lung disease
that I’m gonna talk about, have been the focus of this large animal effort by these national
primate centers. And in the United States, we have seven of
these centers, and one of these centers is at University of California Davis, where I
trained, but also over the years I’ve had opportunities for funding because it is the
only center, primate center, that has facilities for respiratory research and what I mean by
that is they have certain equipment designed for exposure of these large, the primate. So let’s just look at the primate and just
talk about why and why not it might be a good animal model for asthma. One is the airway structure and function is
very similar to the human. Anatomy, the cell biology that is similar. So I just caution all of you, when you’re
looking at your model, if you’re modeling for an animal model, if you’re using that
as a model for human, understand the anatomy of it and molecular biology behind it and
really concentrate, first of all, on understanding the animal that you have for a particular
disease. And that’s what we have tried to do for this
and also the rodent. The models of asthma are similar, more similar
to the humans than in the rodent. For example, the rodent model is often very
short. It’s a short exposure to an allergen, and
you get some information on the response. But the large animal allows you to look at
chronic disease, where you have much more of the phenotype that you see in the human. It’s also, it’s been pointed out before, they’re
better models for lifespan studies, and there’s a tremendous emphasis in respiratory research,
understanding the effects early on, in the unborn, all the way to the aged. And this is why this large-animal model is
a good one, I think. There’s also more heterogeneity in the age
of, we’re gonna see a lot of effort being put into the precision medicine. This is gonna be the future. It’s gonna close lots of gaps, I think, in
translation. And we need to have models where there’s lots
of variation that fits the human condition. So let’s just look at the anatomy between
mouse and monkey. And I point this out because the diagrams
are just of the respiratory tract, the nose, the trachea, and then into the lungs, and
this is a cartoon of the rodent. First of all, you can see that the, not just
the size, but also the anatomy, can be quite different. There’s only, mainly, two large lobes, and
there’s different lobes that are used in the two large lobes in the human. In the rodent, there’s the left lobe and four
other lobes. And so there’s just an anatomical difference. But if you get down to the airways, what we’re
talking about, one major difference is the human has many generations of conducting airways
called respiratory bronchioles. And this is the target, or one of the targets,
for ozone. And this, in the rodent, there are no respiratory
bronchioles. So just anatomically, this model is very different. But if you look at the, this is the, look
at just the size of the epithelium that lines these airways in the large upper conducting
airway, the monkey’s epithelium is very thick and it has lots of mucous cells. And in the mouse, there’s hardly any mucous. That is a different cell type called the serous
cell. And it’s very thin, watery type of mucous. If you get down into the lung, where the conducting
airways meet the alveolar membrane, where you have a gas exchange, there, too, is a
major difference. So there’s much thicker wall of the conducting
airways of the deep lung compared to the rodent. And there’s also large bundles of smooth muscle
is very, a much smaller amount of smooth muscle in these terminal bronchioles in rodents. So why that’s important, especially in respiratory
research and air pollution, is that the pollutant actually targets the respiratory bronchioles. And deep into the lung. And so here are some studies that we did a
long time ago, looking at the effects of ozone on these respiratory bronchioles. So we opened the low, dissected the airways
into the exact same generation of airways in the alveolar, or respiratory bronchioles
and then characterized the response and did that with all the animal models. And with all the animals that we did, we characterized
the epithelia and inflammatory response. And after long-term exposure to ozone, you
can see this thickening and corrugation of the airways that lead to pulmonary dysfunction. But the same changes, I’m not gonna dwell
on this, but also the upper respiratory tract. And so in the human, the monkey is more similar
to the anatomy in a human. It has a, first of all, has a large amount
of respiratory epithelia, like the human, and very little in the way of olfactory. In the rodent, half of the nose is covered
by olfactory epithelia. So again, I’m just emphasizing the anatomy
differences in these two species, and the similarities. And we then went on to characterize all the
epithelial mentions of where the epithelium lie, in the nose all the way down to the conducting
airways in the lung of a primate. And if you look at the response to ozone,
we see there’s lots of respiratory cilia that lines, this is a carpet of cilia looking at
a scanning electron micrograph. This is a normal, after ozone you lose the
cilia in, this is in the upper airways, in the nose of the monkey. And then there’s increase in mucous goblet
cells. And this feature here, I will come back to
this later, but this increase in mucous goblet cells, the thickening in an inflammatory response,
is the key also to asthma and rhinitis that I’ll talk about a little bit later. But then we used other technologies to actually
image the whole, reconstruct the airways. So this is just an example using the nasal
cavity in a non-human primate, these were infant studies. We did MRI imaging and then reconstructed
it, computerized with this 3-D reconstruction, made these computational meshes, took the
pathology and then used more of a metric technique to not only identify the regions in the nose
but also the amount of cells of the different types. So we could overlie this, now, on this model. And then we can simulate the exposure in these
animals and look at predicted areas where the ozone would hit the airways. And this is just illustrating the rat or the
mouse, in the interior part of the nose that would get this damage. And we overlaid that with the pathology and
that was exactly where most of it was. So it was still symmetry where, there are
some areas where you would see the different cell types. So we did another study in the monkey and
then started looking in the rat, and then people picked up on this and this was the
first article that showed the same nasal regions in the human. This was a study that was done in Mexico,
studied by a pathologist Leslie Calderon. And interestingly, in all the species, which
is a rarity, we found all the same effects. Then I took, characterized this in the rat,
over a long period of time, the different concentrations. This just shows the increase in the thickness
of the epithelium that lines the conducting, the nasal airway, and the increase in mucous. So what happens in both the primate and in
the rodent, you have this initial injury by the pollutant, the ozone. That causes an inflammatory response, an increase
in neutrophils, and that led to this metaplastic response. This response is characteristic of asthma
pathology, where you get lots of increase in mucous. But then we started to look at also in mice,
and I just want to show you some epidemiology associations, in just the last few years,
have been shown that there’s association with high ambient concentrations of ozone and the
onset of asthma in a large study that was just conducted in non-atopic, or non-allergic,
Latino children. And also there was a big showing that ozone
causes eosinophilic inflammation and other inflammatory cell types, the eosinophil that’s
often associated with asthma, this is also increased in children. But interestingly, not in children with allergy
but in children without allergy. And then finally, we started to do a lot using
mice after repeated exposure to ozone, we identified eosinophilic rhinitis, nasal epithelial
remodeling, and then better understood type-two inflammation. So let me, in the remaining notes, just show
you what we have found and how we need to carry this back to the large animal model. So two of my students, one a resident, Dr.
Bing, is back in Singapore, and Dr. Kumagi, who was a visiting scientist from Japan, really
did the work to identify the pathogenesis of ozone in these airway injuries and identified
a really important fact and that is they identified the cell type that we believe is actually
the driver of almost all of these changes except for the early epithelial cell death. So all this remodeling that takes places after
nine days of exposure that we saw in the example, increase in eosinophils, mucous, were all
driven by the innate lymphoid cells, a specific type called type-two lymphoid cells. So what are they? These cells are a newly discovered family
of immune cells that do not express the lineage markers of T cells or B cells or dendritic
cells or macrophages. These mirror the phenotypes of T cells, so
in asthma are often here type-two helper cells. But we’re gonna show you that that response
isn’t driven by these T cells in asthma in this asthma phenotype, but it’s driven by
these innate lymphoid cells. And unlike T cells, they do not express the
antigen responses or undergo clonal selection that you would see with adaptive response. They respond quickly and actually release
cytokines that are very very similar, more of the cytokines than what you see in the
T cells. So the ILCs respond to the injured tissue. There are alarmins that are specific ones
or we call it epithelial cytokines that are released and stimulate the ILCs and they produce
cytokines that change this epithelium. So these Ilcs play a role in pathlogy. And there’s a number of them, different types. And they’re actually types that also mimic
the T helper cells. And we’re gonna concentrate on these ILC-twos
because they’ve been associated, in humans and mice, to allergy and to asthma. So what happens is that, we believe, the ozone
damages or signals this airway epithelium, releases these alarmins or these epithelial
cytokines, we believe inter-loop at 33, IL 25, or TSLP are really the main drivers here. They stimulate the innate lymphoid cells. They can also stimulate the other cells, but
in this pathway of adaptive immunity, we just don’t see this in the ozone response. And so what happens is these ILCs can then
release the cytokines, increase the mucous that I showed you earlier, that was shown
in the large-animal model and in the rodent, and this causes non-atopic asthma. So let me demonstrate it. First, the aim was to determine the onset
of ozone-induced eosinophilic rhinitis, in this case. And rhinitis and asthma go hand-in-hand in
their disease. I’m gonna start out in the nose and then end
up in the lungs. So we expose these animals to the point five
part per million ozone for one, two, four, or nine days. We did nasal histopathology, morphology, immunochemistry,
and also RT-PCR. And we found that the lesion was in the anterior
part of the nose, like in the primate, and it was on the lateral surface, very focal
area in the nose. We then conducted, looked at nasal epithelial
thickness but also the inflammatory cells. So I’m gonna focus it, then, on the inflammatory
cells here, the neutrophils and eosinophils. So after day one, this is day one, after a
couple hours and then after 24, and then we’re looking at after I can’t read that here. But this is one day at 24 and then at the
nine days. The key is that the neutrophils are present. It’s a neutrophil archetype one inflammatory
response early, but then it switches, with continuous exposure, to a type-two eosinophilic
response. And this is illustrated here, and we have
increase in epithelial thickness, but we have a switch in inflammation, from neutrophils
to eosinophils. Classically, a classic cell type of asthma. We then went on to look at gene expressions. And that same thing, bottom line is that we
had a switch in the gene profile, from a type-one inflammatory response early after one day,
to after nine days it goes into a type-two response. We then looked, by using genetically-modified
animals, we knocked out, used Rag2 to examine the animals we knocked out the names of all
of the lymphocytes. And that actually eliminated the whole response. So we eliminated, we took out T cells, B cells,
and these innate lymphoid cells, and all the responses, and gene responses as well, in
terms of inflammation, actually all went white, which is quite amazing because this really
strong irritant, pollutant. So that just told us that lymphoid cells are
important. Then we went on to look at innate lymphoid
cells. In this case, what we used is Rag2 animals. And these animals, we were able to show, to
use these animals because they have T immune cells. I’m sorry, they don’t have T or B cells, they
have innate lymphoid cells, while the Rag2 gamma C animals have no innate lymphoid cells. And when we, the animals that have innate
lymphoid cells, wild type or the Rag2, all the lesions come back. And the T responses as well. So in this study we showed that in the nasal
study that innate lymphoid cells were associated with these lesions. When we took them out, the lesions went away. So then we carry that in the lung, same approach. And where we eliminated the innate lymphoid,
or all of the lymphoid cells and some of the animals have just the innate lymphoid cells
and the same exposure to one day or nine days. And just to illustrate here is that with this
animal model we were also demonstrated, we look at the different inflammatory cell types,
the eosinophils that, in the animals with innate lymphoid cells, you actually can have
eosinophilic response, characteristic of asthma, also lymphocytes. But in those without the lymphoid cells that
is all gone, even in the face of continued exposure. And so we also looked at it after one day
and found that that wasn’t the case. So just as we had predicted, is that innate
lymphoid cells aren’t important in the very beginning but when they go to the asthma-like
phenotype, they’re the major drivers. So this also shows the mucous cells in the
airways were present in those with the innate lymphoid cells and not with the ones that
didn’t have them. And we further went on, then, to look at different
gene expressions for mucous and for inflammation in the same plane, as demonstrated. So we used a we also wanted to use flow cytometry
to understand whether or not these animals had increased numbers of ILCs. And so the animals, the Rag2 animals that
have ILCs but not T or B cells, when you’re exposed to ozone there’s indication that it
may increase, but we didn’t really demonstrate it. We did demonstrate there were no ILCs in the
lungs of the animals we genetically modified. But then we went on to actually treat the
ILCs with an antibody that taps out the lymphoid cells. So we knocked out the innate lymphoid cells. We only take out less than 2% of all the cells
in the lungs, but we were able to knock them out in the phenotype that increased the eosinophils
that we saw, that was eliminated, as well as the mucous in the airway. So we think that these cells are very very
important in the response to air pollutants. And they are important in this non-atopic
asthma. And this was used, we demonstrated this in
the mouse. But I think the key, after I showed you all
the things where there’s differences between the large animal and the rodent, that really
needs to be carried back into the primate model. And this now is one of the areas of focus
by some of the researchers now at UC Davis. The real thing is the treatment, though, the
translation of that. And so one area of translation that might
be very helpful for asthma is actually to look at how to deplete or to stop the effects
of these innate lymphoid cells. And it has to go into a model that mimics
the human condition. There are some pharmaceutical companies that
are actively exploring that, these alarmins that have been demonstrated. So we were the first to demonstrate this in
the mouse. There’s two other labs that have verified
that now. But I think the key is here again, these large-animal
models, before we can translate it into prevention or treatment. So I close here with some suggested readings. And those readings are really, I think, if
you haven’t read the book yet, Thank You for Being Late: An Optimistic Guide for Thriving
in the Age of Acceleration. I think you’ll find this extremely great book. It touches on a lot of areas of where we have
lost boundaries and now we have to survive and add to this age of big data and how they
translate it. We are gonna have to work together on that. We have to take time to really really think
about what we’re doing, that we’re not just generating data that the bioscience that we
do is not lost, kinda lost in translation. And so this is also a good book, by Richard
Barker, on that. If you’re interested in laboratory primates,
there are a couple chapters in this one, is a good one, probably, to start. Right this one is just an example, certainly
you think that maybe an approach is to looking at farm animals or other large animals and
beasts. So thanks for your time.

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