Immune 54: Asthma and aging

From Microbe T V, this is Immune, episode number fifty four, recorded on march thirty first, twenty twenty one. I'm Vincent Raccaniello, and you're listening to the podcast on the body's defenders against disease. Joining me today from Ithaca, New York, Cindy Leifer. Hi again, how are you? Very well. How's the weather? You have cold or warm up there? It's warm today. We are almost sixty degrees and then it's gonna snow again and our ski resort is probably gonna be open this weekend.

Are you serious? You gotta be able to ski? Holy cow. I I I probably won't, but yeah. It's insane. Yeah. Uh from Durham, North Carolina, Steph Langle. Hi, hi, great to be here. Uh back again. Thanks all for for doing a great job last month while I had some daycare shutdowns. Uh but good to be back. And from Madison, New Jersey, Brianne Barker.

Hi, great to see everyone. I'm excited to talk about another immunology paper today, second one I've talked to Vincent about this week. Um so that'll be fun. And it's warm here too. It's almost seventy. Ah yeah, I was gonna It's so weird. It's warmer there than here. Oh really? Yeah, yeah. I figured you would be the warmest. It's raining outside, I see, but It's raining and incredibly humid, but also sixty nine degrees. Okay. Oh you choose your poison when you like complain about.

So Steph, as you know on a recent twiz we were talking about you and Linda I know. I enjoyed that. I said you know, I I said you were in Iowa and so you corrected me and then I got an email from someone and said, No, they're not in Iowa. Oh that's so funny. I said this. It's all the same, isn't it? To New York Island. Because when I heard you say that I was like, Man, I don't think so. My remote.

But yeah, it's like But my reference was I thought Stephanie was from a sports school and that I've talked about sports with her, so I thought it was a possibility. Oh, Iowa people would be very mad at you if you I but I was specifically felt like uh I'd talk to you about, you know, Duke versus Ohio State sports. Yeah. By only point of reference.

Yes. Well, you know, it's funny'cause when I I almost didn't text you, Vincent, but I said, Okay, this is my strategy. I'm gonna text you how much I liked the episode, which I did loved it. I'm I'm gonna say thanks for for bringing up my you know, my past work, which was awesome. And then the next text I said, But by the way, it's another four litter state in the Midwest. It's Ohio.

That's a good strategy. Instead of saying you screwed up initially, which would be which would be with resistance, right? You were nice and that softens the person up. What a brilliant career you're gonna have. That's really cool. Well yeah, we do have the New Yorkers view of the rest of the country. I'm sorry, I have no problem with New York State. I know where New Jersey is, but Iowa. Sorry about that. No, it's fine. We even v went to Ohio not too long ago, right?

Well that's the thing for the Ohio State people, you did do a talk there, so but both schools are great, of course. Good virology and immunology going on in both.

Well, Steph, in reward for missing last episode today, you get to do the paper. Yes, yes. Um so uh the paper that I'm gonna be talking about today. I'll I'll tell you the title, the authors, where they're from. So the title's Age Dependent Reduction in Asthmatic Pathology Through Reprogramming of Postviral Inflammatory Responses. So it's a little bit in here for everyone.

uh in regards to so we got the viral component, we're gonna talk about macrophages, um and and pediatric m animal models, which I love. So the authors are first author, Guy Hazan or Hazan. And then the last author, Jeffrey Haspel, and a bunch in the middle. And all of these authors are from, I'm pretty sure all, Wash You. Am I correct? I think so, ¿ya?

all look like they're from Washington University School of Medicine in St. Louis. Um and I it's good to know where they are because in I think the first couple of paragraphs they talk about some clinical data and they say in our area I'm like, well I don't know where your area is, so had to look. Lois is what they're talking about.

Okay, so this is in the Journal of Immunology and I like the Journal of Immunology a lot. I like it like I like the Journal of Virology. These are association journals and I think they bring us, you know, it's not gonna be like your jam-packed cell nature science paper where there's 50 supplemental figures and it tells this huge full story, but it gives us some interesting, interesting windows into um immunological concepts.

It also offers opportunities for us to talk about experimental design, what we liked, what we didn't like, and of course, as we do on these shows, you know, all of this is is is said in uh of course we enjoyed the paper, but then, you know, there's always there's always things to improve. So we'll talk about that too. I and I also love Ste Stephanie that you mentioned sort of the the you know, the scope, um, even though it is an eleven figure paper.

That was I was gonna ask, is this the longest JI paper that you've seen? I kept going. I've seen the longest JI paper I've seen. I I didn't know they allowed past eight figures. Yeah, I thought there was a limitation. Like a supplement. You could put that in supplementals. Yeah, I was surprised too. Um but A lot of this I g I there's a something I don't know what things I could think could have been supplemental, but uh that's a that's a good good point. What did you think, Brian?

Yeah, go ahead. Yeah, I w I would agree. I think that there were a lot of really interesting observations made here. Um that Provided fruit for f future follow-up, and I also saw some things that I thought probably could have been supplemental. Um, I think this is the longest DI paper I've seen.

Yes. Yes. Um so uh what we're gonna talk about today broadly is asthma. particularly pediatric asthma. Um and and I'll define asthma. So with with they their first sentence in their paper, asthma is a common lung disease characterized by paroxysms of airway obstruction, airway hyper responsiveness and inflammation. And you know, if you if you haven't

Gone to medical school and you don't know what paroxysms are, uh, it's a sudden reoccurrence or attack of a disease. So it's like a sudden worsening of symptoms. which is characteristic of asthma attacks, right? Your your your lungs, the l lobules are are constricting and it's causing reduced blood flow and oxygen. Um and and so This disease has a genetic susceptibility. And so there are individuals who have genetic contributions to asthma susceptibility. And that that's has been shown through

family studies, heritability studies, twin studies. Um and it also is exacerbated by things like infectious agents, viruses like we'll talk about, allergens, and the allergens can range from Pollen, mold, animal dander, insect parts, some chemicals. Um and it's also exacerbated through recurrent exposures. So exposures of these agents over time.

at certain age groups seem to exacerbate it. But what's interesting and and there was some work done at Ohio State that in Amish communities, rural communities, farming communities, that the levels or the risk of asthma is actually lower in those children, particularly when mothers on farms th there is this um hypothesis that low levels of recurrent antigen exposure, particularly endotoxins, could

prime the system away from this and we'll talk about what this means a TH two driven eosinophilic uh uh response in the lungs. And that's interesting, right? Because endotoxins are going to, you know, it's LPS, that's E. coli. stimulate a different toll like receptor um on the surface of cells than something like a viral infection, an allergen. And so th there are there are things that can reduce your risk of asthma. Um having pets, being around livestock is is one of them.

question. Yeah. Um so we so you you said this really nicely that when you have infections that drive more of a TH one type of response, that makes logical sense of biasing away from TH two driven diseases. and and asthma's one of those. But what about there's also the hygiene hypothesis where we've had better clean and w you know, water and so forth, so we've reduced our Um parasitic exposure, which is TH2. And that also seems to correlate with lower levels.

of TH two driven diseases. And I've kind of always said, well, that's'cause they're they're uh teenagers that tend to get in trouble and you occupy them with something like a parasite, they're not gonna cause trouble another way. But the reality is it seems like either one of those two could reduce the amount of T two driven disease pathology. And do you have thoughts on that?

Oh Yeah. Oh d Brianne, we're you're gonna say you go ahead. Oh so um I've seen sort of two models uh that address this um and one of them kinda helps me understand um this is that In the case of the parasitic infection, you could imagine that um in a population where there are many people who uh have chronic parasite infections, that the overall level of immune responsiveness might be a bit lower in the population, sort of the mean immune responsiveness. Everyone's a little immunosuppressed.

Um and so there are fewer people at the high tail who are making very excess immune responses. Excuse me. While in a population that does not have that immunosuppressed um phenotype, there are going to be more people who are sort of at the level that is enough to have an excess response. Um so I've seen some really uh nice kind of figures trying to depict that and I think that that has helped me understand exactly the question that you posed.

Interesting. Do you also think it could go it could relate to what I mentioned about lower levels of Exposure early in life. And so so the the farm example, high endotoxin levels, LPS from animal feces or whatever.

uh dog dander. Could that also explain that if you have a a if your response is generally type two, that there's a there's a reprogramming that doesn't there's not a hyper responsiveness. And I guess it it could go back brand to what you're saying, but It seems to be in these cases where

pediatric patients or individuals they they're dipping into a TH two. It's like they're at a T H one and then they can't handle the TH two and it and it and it kind of accumulates upon itself to to cause this attack. I I don't know. I think maybe we could be saying the same thing, but something about You know, priming the immune system and keeping a general TH2 environment could could be the reason.

Sounds like we could be saying similar things. Could be, yes. Um one also other thing that I think was really interesting when I was thinking about some of the background information that they were discussing here. is that they talk a little bit about um differences in asthma with age um and sort of what is seen in uh pediatric asthma and what's seen with asthma of different age groups.

Um and you know, I am not a physician. I have not gone to medical school. Um, but I think that I have always understood that adult onset asthma and pediatric asthma were different things. Um and differed a bit in severity. And so I think that um and when people have asked me to go into more detail on that. I've said, Well, yeah, I think that's how it goes. And I can hand wave some reasons, uh, immunologically why I think those would be different, but I haven't really seen solid data. On that.

Um again I could come up with a little mechanism if you someone really wanted me to. G get you a dry race board. Exactly. But I I liked the idea of this um starting to address sort of the idea of some of these disease states that are seen in different age populations may actually be functioning different.

Yeah, yeah. And actually I'm glad you said that functioning differently. Because I think I do want to take a step back just a couple of minutes and reframe um the way maybe we talk about teach to uh type immunity helmet infections. I think because Immunos. I don't want I think immunosuppression is maybe the wrong picture of TH2 because I think it's more.

People who have more TH two, who who maybe can better fight helmet infections, they actually uh in some cases not if they're HIV positive, are less likely to get cancer. Though th there are other diseases that they're less likely to get.

than those who don't. So maybe a way to think about it is you just, depending on your environment, you you're skewed either way, but one, type two, actually could have benefits for this thing compared to this thing. So I think I always I hesitate with immunosuppression. concept unless people are like on immunosuppressive drugs. That makes sense.

Coming back to what you were saying, Brianne, about adults versus kids having different types of asthma, I did think that that there's a l a lot of kids who actually have asthma that's triggered by various different things, but a lot of it I thought and I I could have thought wrongly, a lot of it is infection induced asthma.

Which is sort of related to this. So I think kids are frequently hospitalized after they're already sick and they develop bronchiolitis and and some asthma associated secole from that. Is that do would you agree with that? Yeah, I think that that's true and I think that that predisposition is specific to kids in many cases that in some ways they sort of grow out of it.

And I think that's sort of what they're trying to address here is what what might the viral infections be doing and how might that change over time? Like how might their response to the viral infection change over time and that might change their predisposition to these asthmatic events. And so that's that's why I was wondering because if if they're framing it this way, is that the same as someone who is not infected with anything that we know of? having an acute asthma attack.

from some kind of they breathed in something and or we just don't know why, but they have this attack. Is that the same thing? And is that also impacted by this? That's sort of how I was thinking about that. Yeah, I I feel like that would be really hard to answer. Yeah. Because you would if we think about some of these infections that we're talking about, um, many of them seem to be common and you could imagine that in a lot of cases with that acute asthma attack.

it would be hard to know if there had whether or not there had been a predisposing viral infection that maybe had gone di undiagnosed earlier. Um so I think it would be pretty tricky uh to fully address that question. It's a really interesting question though. A a vast majority of these are associated with rhinovirus infections. And um often what happens is the kid gets

infection they s they start with a wheezing illness which then progresses to asthma. What's interesting with the rhinovirus is that only certain types are associated with wheezing illness and asthma. types A and C and the Bs don't do it, which is very interesting. And not understood why, but so there's some virus component as well. Interesting. And I'm s do do rhinos infect mice? Is there a mouse model that you could st study that? It's funny that you should say that. Oh okay.

There is there is some there is a a mouse in the literature but You know, the virus doesn't reproduce. You can put virus into the respiratory tract and it doesn't reproduce, but So I always thought we years ago we tried to adapt the virus to mice. And it's funny because today I just got an email for someone who would like that virus to do some drug screens in mice. And it's like we threw it out years ago. Nobody wanted to do that. But uh I don't think there is a good mouse model.

Interesting. Okay. But you know, the thing is the virus b replicates very poorly even in humans and it doesn't kill cells. It doesn't kill cells. So some kind of sensing issue is going on with asthma, right? I bet it is. Oh yeah. Okay. Interesting. Well, uh you know, Brianne and and Cindy, what you guys are talking about is just a really great setup because they do give us

some stats. And so'cause they they want to bring us into this uh this phenomenon that There roughly fifty percent of the children diagnosed with asthma before the age six will experience remission of their symptoms by early adulthood and then twenty to twenty five percent of the cases will achieve resolution of their airway hyper responsiveness.

And so the exacerbation rates really are they they fall as children age and then they they plateau and they reach adulthood. And so what is this how how is asthma aging out. Uh and and I that's not understood. And and one of the things that they they they talk about is that the seasonality of asthma exacerbations in children. um corresponds to viral disease. And so y you can find that asthma in uh emergency department

visits. They start when school starts, so they start in the fall and they peak during the seasonal flu, cold and flu, and then they go back down in the spring and summer. And and so that's another indication that viruses probably pay that play this larger maybe an outsized role in particularly in children. And and why why is that?

And one of the things that they they present to us. So so the first figure, the first set of figures that they dive into is we have an experiment that COVID nineteen gave us. And and that experiment was what happens when we implement measures that do not allow for the transmission of childhood viruses, and that is COVID-19 associated lockdowns, which are, you know, masking, social distancing, shutting down schools, shutting down uh parks.

And what does that do to the rates of emergency department visits due to severe asthma? And i it w it's amazing and there's a lot of anecdotal evidence that I had heard from my past postdoc mentor, Sally Permar, that they they go away. That post lockdown, the seasonality and the in the the increase, the huge increase in emergency department visits.

and young children went away. I'm sorry, I shouldn't say that it went away, but the cases were the same it's the same amount of visits per year as older age groups. And that's pretty incredible. I mean, it's a sub it's a seventy percent reduction. Yeah. And and I they also talk about how the seasonality, as you mentioned, also went away. So instead of seeing different numbers in different months of the year.

Um that this sort of background number, the number they see in older and younger children, also was this that same background number throughout different parts of the year. Right. Right. And and so it implies this potentially developmental process that mutes the host response to seasonal triggers in children. Um and and that They so they one thing they mentioned is that this is

And these were antiviral measures. And so I I I would like to tweak that because masking social distancing, while of course the end result is reduction and transmission of viruses, it it doesn't uh remove the contributions from allergens. If we treated everyone with an antiviral, that would be direct evidence of an antiviral effect. I think that we can try to remember that these kids aren't going to schools and schools

have large amounts of allergens, right? Especially in older school buildings. There's mold, there's there's cockroach dander, you know. So it it it wouldn't necessarily all be attributable to viruses, but for sure a larger a large portion.

One of the things that they said also was just the lockdowns in general reduced the overall respiratory virus positivity rate, right? So that's like one of those things like everybody saying, Oh, the lockdowns don't matter, people are still getting sick, et cetera, et cetera. But I mean they just

Their testing shows clearly that these measures actually reduce transmission, which makes perfect sense and is the the the basis behind health policies and things like that, even though people want to believe that's not true, it really is. Yeah, I I was excited that they cited their specific data on that.

um so that I could sort of see a data source and I um made note of some of the uh references that they actually uh used here um in talking about that um to go back and take a look at some of them um for uh my future use uh in classes because I was in the same way being like, Oh, this maybe these could be the perfect data to to show this um to those who need it to be shown. What's interesting though is that As far as I remember, rhinovirus infections did not abate.

I remember Amy saying this multiple times. We looked at a paper on Twiv a while ago with some data on that. Okay, that's interesting. Um I don't know what that suggests. Um I don't either. I mean she s m suggested maybe the transmission is different. And so but if you're locked down you're staying home, right? So I don't know because if we think about it.

Yeah. And and so I guess all right, what you're saying, Vincent, and kind of a a lot of this is, you know, we're looking at broad trends and trying to speculate there's probably other contributions. And and the other thing to mention, I mean, there so there the the brackets of time that so the pre lockdown was a four year average.

And the post lockdown was one year. And so your your confidence intervals in the pre lockdown average are gonna be tighter than the post just because there's more there's more kids. Right. And so you can even see that in the data where There are differences in emergency department visits, for example, in six year olds.

It's at ten visits per year and then it goes back up in seven year olds and then back down in eight and nine year olds. So I think what this represents is it's not that there's probably biological relevance why your six year old has less emergency department business in your seven year old, but it's just there are less kids to make that mean more well smooth.

That's in the allergy responses, which weren't different. Um and so you know, that's one thing that's really unique about this is that the asthma visits Huh the asthma visits have gone away, but the visits for other types of allergic responses really haven't changed, um is how is how I was looking at this. Yeah, it's interesting though because again I I mean Those confidence intervals don't interlap uh overlap. So I was wondering like it kinda looks Real term.

Were there really are not any s any confidence intervals on the one year post lockdown'cause it's just one year, right? Right. So I think it's it is it there are some caveats with this paper. It's it's provocative, it has some really interesting things, but I think there are some limitations. Sure. And that's one of them. I I think it's a good idea. In their favor on the asthma part. I agree with you completely on the allergy part because I spent a lot of time squinting at that graph.

And looking at a couple of different points and thinking about what would happen if those points changed. On the asthma front. Lockdown measures. It's pretty it's pretty clear and Yeah. I have... Yeah. have not been to Saint Louis in a very long time. Um But I wouldn't say that all of the lockdown measures that happened over what these years were 100%. You were that is very true. And with less than a hundred percent uh uh mitigation measures, that's a huge difference in asthma case numbers.

Yes, you're right. You're right. So you're right. I think that's a great point. So so they're saying maybe the allergic reactions are not different, but the asthma is hard to say about allergic reactions, but you can't argue with that asthma data.

Thanks. So here's a little clarification on the rhino. I'm looking at a study where they summarize Current non pharmaceutical interventions were not effective in preventing rhinovirus infections of children, although they were effective against influenza and RSV.

The rhinal de infections decreased during the initial lockdown with school closures, but as soon as the restrictions were eased, detections returned to near normal level, and this is in multiple countries. Unchanged rhinovirus detection rates during social restrictions. Huh. Could that mean that rhinoviruses are even they're even more fit to transmit? Well, I don't know. It's a good question. I mean th one of the ideas is that there's more of a role for finger

contact transmission than for SARS-CoV-2 and other viruses. Right. In and in fact in transmission studies with volunteers and rhinoviruses, what they do is they put'em in a room and they have one infected volunteer and they play cards. And the cards transmit the virus. Interesting. One person sitting there who's not playing cards, so they're not touching anything, they don't get infected. That's interesting It's not on cards.

Right. Yeah. I I remember when we looked at the data on TWIV, um, feeling like there was maybe something to enveloped versus non enveloped viruses. um looking different, but I'm not sure that there was necessarily enough data to really uh make that as a claim. Yeah. Yeah. Well I think it's interesting'cause I don't know about you guys but w so we're

We don't have a mask mandate anymore, although it's still recommended, which uh you've probably seen in the news. Cornell makes news all the time now. I don't know. It's really great. But um I I am noticing that uh everyone is less um worried about fixing their mask.

So there's a lot there's a lot of masks touch it's constantly pushing up, pushing up, pushing up, pushing up, adjusting, adjusting, adjusting. And so even if you're wearing a mask, if you're if you like you said, if it's more transmissible by touch and surface, then you still might get it more, you know, touching eyes, wiping eyes. Because I I think very early on, even when we were being told the masks were working, it's less transmissible, like people

If you remember from COVID nineteen, people were spraying down their ev all their groceries and everything they every piece of mail. I mean it was crazy, right? Then it was like that's not the way you're getting it. You're getting it from breathing to wear a mask. Right. So people were less worried about touching things. But yeah, I mean the different transmission might be Um Of RSV or or rhinovirus might be related to that.

And I also think during lockdown someone in the house went out at some point to get something. Right. Thank you. Wipe and both. I don't understand the asthma decrease. It must mean that there are multiple viruses involved. Right. So I I agree with you. I think that it it maybe the contribution from rhinovirus also involves

secondary viral infections. And and and they do talk about how having a pre exposure to to multiple viruses can contribute in particularly genetically predisposed individuals. So Or it could be that I mean I agree, I think you could have multiple or it could simply be you remove one and the others take its place.

Yeah, yeah. Right. So so that i w was an interesting uh piece of data and then some anecdotes that we've heard um from colleagues, but they wanted to probe this further. And so what did we do? We built an animal model, we develop an animal model. of asthma or at least the manifestations of asthma, um which in their case they're modeling post infectious lung remodeling. And so what that means is things called mucous cell metaplasia.

And metaplasia is when cells pop up where they weren't before. And so these are mucus producing cells, usually goblet cells. So you see an increase in mucus production from goblet cells after In this post lung remodeling, you would have an increase in type two inflammation based on inflammatory cycle type two cytokines.

IL four, IL thirteen, IL five, I thirty three, and then um they talk about M two macrophages, which I'll touch upon in a bit. Um, and then this airway hyper responsiveness, which how they measure this in mice is they they they they have this oh I looked it up but uh they have this system where they they basically can intubate mice and inject metacoline. Is that the pronunciation am I saying? I think so.

Metacoline and it is a parasympathetic chemical that binds to receptor that causes you to cough, and it's a way of of of assessing the um restriction in the lungs and and the the the r yeah like how quickly do your lungs respond to that chemical and that's that's a measure of airway hyper responsiveness. Um now when they they show us their timeline, so what they wanted to do was they wanted to give the mice a viral infection.

And they pick Sendivirus or Murine Parainfluenza virus that's in the parom para mixoverdae family. And Vincent, I'd be interested to hear your thoughts on this, but from what I understand that this is a a virus that can m it persists long term and um it the lung pathology persists long term so they can model like this acute phase of infection but then chronic lung changes are happening. What they they've picked day 49, they don't really tell us what's

relev you know what what's significant about day forty nine. I'm sure there were some other studies to demonstrate that day forty nine is relevant. Um So it's it's a model of respiratory infection that can induce chronic lung changes.

Yeah, that's right. Um and so so they they're showing us their model and they're demonstrating that um they airway there's an airway mucus gene called is it muc five A or muc five A C I always say mukfire. So okay, muck five A five A C. Um which is a mucus related gene. It's one way to measure it. It's higher in young mice compared to older mice.

That was also true for this particular staining of mucus associated glycoproteins. So they they in the juvenile mice there's uh higher levels of the PAS staining after the sendivirus. infection, but not in the mature mice. And so they're defining young and older mice as six week mice as the younger and eleven month mice as the older mouse.

Um and they are also looking at things like IL thirteen, which is our type two cytokine, and they see an increase at day forty nine. So this is this is more of a chronic uh time point higher in the juvenile mice only after what they uh sendivirus and uh particularly is seems dose dependent with higher levels of sendivirus. Oh sorry. Earlier in the paper they tried to um tell us uh how the mouth these ages of the mice correlate to human ages.

So uh they mention they say that um what is it? The four week old mouse they they say is uh equivalent to uh between two a two year old and eight year old human. Um they say a sixteen month old mouse is akin to late middle age in humans. Um I suppose one can make their own decision about what that means. And in the Like fifty. And then oh see, I thought that was the citation. Oh maybe. Um and then put they also talk about three month old, which they said is early adulthood.

Oh, that's funny that you say that because I assume that that was the age range. I'm like, that's weird. It's two year age range, but you're right, it's probably I ru 'Cause I was like middle old age. I I don't know if it's just Forty eight to fifty years old. Analogous to early adulthood in humans, forty nine. I thought It's like that's what they were saying. Okay. Good catch, Brianne. That's funny.

Yeah. So yes, so they they are kind of trying to model uh you know, and it's hard in all these animal studies. So we try to do this in monkeys we try to say, okay, an infant monkey, what does that represent as it ages in comparison to to humans and and we can do this in mice, but of course it's hard because mice age very quick. I mean, they they grow up very quickly. They're adults.

by eleven months of age. So take it with, you know, whatever grain of salt you need for that. But for sure, it, you know, they're they're trying to model the range over the course of the life of the mouse.

So I was thinking about this and It's it's funny because I t I ta I was just talking with um people in my immunology class about how to how to look at data and it's it's higher in one group than in the other, right? And so the question is why is it higher? And one could say These uh mucus production and muck five and these cytokines and things are higher in the juvenile because they're over responding.

But one could also say that they're lower in the mature because they're failing to respond, right? Because we always talk about aging and how aging influences immune response. And so it's saying the same thing, sort of, except it's really interpreting it a different way, right? Because they want us to believe that the juveniles are actually hyper responsive. But what is normo responsive, right? Where what

Which one is high or low? Like is there one in the middle or does it flip at a certain point and how does that relate to to protection and pathology and things. I don't I I don't have an answer for that, but it's just an interesting way to think about it. Yeah, but right. So and I I guess it is in the context of the question you're asking, because you're right, this hyper responsiveness. Um at least uh some in data that we're seeing with SARS CoV two.

children have maybe higher levels of things like interferon than than adults. And so in that context, that means better protection because that's clinically what we're seeing. So yeah, it is you do have to especially with these age studies, think about, okay, what what is the context and are we potentially like putting something on this age group um without the data. You know what I'm saying? Like we're we're saying these are higher or lower, but is that really to work?

I mean, is this just because they're better at fighting the virus? And this is an outcome of that. Right. Which we will we will touch upon because there were some interesting statistical questions I had about some of that some of that data.'Cause that's a good point. What with the f the what this figure is showing us is there they want to show us that

this this chronic time point, day 49, where it's it's a post acute infection that there is this exacerbation in juveniles. But what happens in the acute phase, and so in a couple of figures, we'll learn a little bit more about that. One thing is missing here is virus titers. Yeah, yeah. We'll look at that later, but they did not look that easier.

Experiment. Um They say the peak viral load is between days three and five, but I would like to see that because how do you know it was in your experiment, right? Right. How do you know that they cleared at the same with the same kinetic? Exactly.

Right. And and what we could do is we could jump to figure five, and this is a an opportunity of improvement that I would suggest to this paper if if I was a reviewer, is that some of the data does jump around. So that That question is a pertinent question because if the juveniles just are less good, quote unquote, you know, less effective at reducing viral titers, then that could lead to more lung damage and then potentially you you know have more chronic

Uh chronic effects. But if you so figure five, they're showing us that after sendivirus infection, that the viral is a very important thing. Uh RNA ex levels are not different Except for day eight, which they are. Don't you guys see that too? They say they weren't different at all, but they actually that that's different. Well i it also it's at eight and twelve. Right. Don't care about RNA. It's infectious virus that matters, right? Correct. Oh, I know.

An opportunity for doing that would have been really v helpful here. I mean you can even say that it's too hard to do the infectivity assay, right? You don't have to do it at B S L three like you would have to do for SARS CoV. Do you can do this and there's no reason to do a P C R. Absolute this is totally uninformative this panel B, because it could be pieces of R and A and slightly different pieces. So I would yeah, they need to look at infectivity.

Yes. and the number of cells it infects in the mouse. They did. And that was something interesting that although I mean they say in the paper that vi viral RNA levels weren't different, even though they are, if you look at day eight and twelve, and maybe they didn't want to talk about it because the trend flooded went from higher in mature at day eight and then higher in juveniles at day twelve and maybe they're like, I don't even that doesn't

Maybe that's not biologically relevant. I I don't know w why that was um not brought up, but they do show you that when they stain for cells in both the juvenile and mature animals, that there seem to be higher increase in the uh amounts of SEV or cendivirus positive airway cells, that the frequency is higher. But they're showing us, of course, one representative image. Um I guess they do they do calculate it with kind of wide error bars.

I I could believe that though. I mean I think I believe the data um that the number of air infected airway cells is higher. That that could contribute. If you have more infected airway cells, you could have higher local damage. Right. But it does they do suggest that they both clear the viral infection by twelve days. So that's helpful for their model because then at forty nine days you shouldn't have any virus left. Right. Yeah. All right. True.

Right. Yes. Let there's little RNA and there's no more infected cells in the lung. But you're right, they should have done that.

No that's probably reflected in the reviewers, it probably immunology reviewers who wouldn't necessarily Want that plaque assay. It wouldn't have made it in journal virology, but it made it in journal immunology,'cause the immunologists don't care as much. And like PCR is fine. Yeah, right.

And then I I did have another question. So they did look at the weight of the animals and they didn't see differences between these animals until they got to day twelve, where there was more okay, so the they defined the weight index. is it's the weight loss indexed to starting weight. So is this suggesting that a higher number means more weight loss? Or weight gain. I thought it was a weight game, but I I also thought it was weight gain because I expected the juveniles to gain weight as they

Yes. Right. So that's what we could okay, great. I think it's just because the way that they state it in figure in in the figure is it says weight loss index to starting with but I agree with you. I think it's higher in juveniles, which we would expect. Juveniles are going to gain more weight because they're growing. So But they did say there's no difference and there is a difference.

Right. So I was like, is there no are you saying there's no difference during the acute phase, which is true, but in fact there is a difference later on. So and and this actually this would have benefited from non infected controls. What are mice what is their weight gain when you don't infect them? Yeah, I mean if you if you don't in fact if you have a a f four to six week old mouse it's just gonna go sh

Because they grow really fast. Yeah. They wouldn't they so in this one, when they're infected, they go down first and then once they clear start to clear the infection, they start to l to gain weight like they normally would. Right. So that uh we we hop down to to kind of the that figure because it wasn't included when talking about the this late time point. So I guess what we could what we could summarize from from this first figure is okay, maybe there's not huge differences in

viral RNA, although debated, no plaque assay. Um, there is higher amounts of infected cells and then later at day forty nine, there's there's a clear difference in the amount of um muc air resistance, mucus gene expression, et cetera.

So they wanted they next asked, Well, is this attributable is this phenomenon, can you see this in other viral infections? Because Can we can we say one more thing about this? Because I found this was really interesting. So they were really careful to say, well, we checked male and female mice. Right,'cause that's that's always what everybody asks. You know, did you do it in male and female mice? Is it the same? And I even put quotes'cause I wrote they said the age effect is sex independence.

Which is true. So, you know, there is an effect by age in males, and there's an effect by age in females, but they completely Lee. didn't comment at all on the fact that the female mice have way, way higher levels of MUC5 AC versus males. So clear and and I found this fascinating because A lot of TH two driven diseases or all preference and it's usually females

Um and I think but I'm not a hundred percent sure and I should've looked this up, asthma is as well. Is there a female preference for asthma? Do you know? I don't remember it being one of the big standouts, but I can only think of the ones that are like super dramatic like lupus. Um So I think it's not that dramatic, but I think there is a increase.

Cupus is a is a huge one, but also for viral infections, don't females tend to respond more robustly? Yes. And so that fits with this as well. Yeah. So I just I I was just surprised'cause it's not a little difference, it's a big difference. Yeah. Yeah.

Um, the the females are way more responsive. And so I just thought that that was another they didn't even comment on that. They just said the age effect is sex independent, which it is, you know, there's a difference, but it just felt like It would've been it would have been interesting, yeah, to Here's one article which says in children Boys have more asthma than girls and and adults women have more asthma than men. Interesting. Well they're just French and old have more

Are you looking at figure two I is that what you're yeah. Yep, I see. Yep, okay. Yeah, they're saying that the younger females have higher Levels, but um they don't have the unin so here's what they don't have. They don't have uninfected young female mice. Right. So we don't know what the baseline levels are. They don't have any uninfected female mice. They only have They have uninfected male mice.

Right. So it's kinda like, oh well, that could just be uh yeah, I don't know. It's hard to say what that means. Right. Yeah. But it but I do agree, we are seeing a lot of data on sex differences and so Th there would be uh evidence for females having higher responsiveness. Yeah. But you're right, that is something so I d I did remember reading that now that the boys are higher uh as I'm young. Bye. So um

Okay, so then they wanted to say, well, what if could do we see this similar trend if we infect them with influenza? So they did r kind of the same experiment, except their analysis of lung remodeling for influenza was at twenty one days? And again like it's kind of

a long that that's a good amount of time before forty nine days. And so I'd just be interested in why twenty one. Is it because they didn't see differences at 49? So they're like, uh, we'll do it at twenty one. But either way, they did see Increased airway resistance in the juveniles. increase staining for glycoproteins, mucus associated proteins, increase in mUC five AC. Um so, you know, they're suggesting that this is also seen after influenza infection, but

You know, there's a lot more that could have been probed. They did look at relative uh RNA expression, not infectious virus, and and they only looked at it one day. So it's I I I see what they're trying to d do because they're trying to relate it to one, a human pathogen, and two is this

Like let's see if this is not just Sendai specific that this is any virus. But I don't know if we can conc conclude that necessarily from this figure. And they don't really do much more with the influenza. So I you know, they I guess it's to say at twenty one days juveniles have higher levels of air ray resistance. We could say that.

Yeah. I think I think this was important to so that show that it's not an a unique thing to send divirus, but that's more general. Different respiratory viruses. Okay, so so then they go into uh you know, I th the presentation maybe I I would have waited and this could have maybe gone into supplemental because they asked the question of if we you know, microbiome differences can impact

Allergy related responses, immune responses in general. So if we wipe out the microbiota with a broad spectrum antibiotic, do you see differences? And, you know, um they the antibiotic did a great job at reducing uh the microbes. But it didn't change the the differences. I mean that you still saw increased aerosistance in the juvenile animals after sendivirus infection, um, compared to the mature

mature mice. And and they looked at gene expression and and they didn't the they didn't see a change in that trend. So It it I don't think it concludes that microbiota doesn't impact things, but I think if you wipe it out completely, the trend is still there. Well, I I think their data show the the microbiome does have an effect. They and they also don't talk about that. This is like another thing where the data are there and they just kind of ignored it. So Do you mean...

So so in the one figure they're looking at the airway resistance. Okay. And they plotted their control mice and their microbiome depleted mice on two separate graphs with different axes. And if you look The the microbiome depleted ones actually have higher Yeah. Resistance. So it's saying that they are hyper responsive. So just depleting the microbiome. is changing. Oh I see what you're saying. Yeah, right. Area responsiveness. I didn't catch the but I don't know if they're

PBS treated mice. U Oh actually I I should say the PBS treated mice were both right around six. But the infected juveniles were more like fourteen in the microbiome depleted versus eight in the control. Yeah. So to me that's a difference. And they they look they viewed it and analyzed it in a different way and they kind of ignored that difference. Yeah. I wonder though with the error bars if they would be different. I I'm just trying to cross my eyes over. No, why did they do that?

Yeah. I I so my question is could do you think you could conclude from this that the microbiome effect is one effect, but there is a separate age effect. Um because I think that, you know, when we talked about this at the beginning, um, we mentioned hygiene hypothesis and the environmental exposures and things like that. And so, you know, what I would take from this is

It's not just those microbiome differences. There is something intrinsic to age, even if you erase the microbiome or make the microbiome the same. And so that's as far as I would have wanted to take these data. is just saying there's something besides the microbiome. And because I agree with you, the microbiome is clearly doing something. Oh yeah. And more in in the in one than in the other, the eight. They're age related.

And I guess that that's why they didn't graph it on the same graph, because they were asking the question of did it remove the trend of, you know, the difference of seeing juveniles higher than mature and not within the juveniles having a difference, do you know what I mean? Because it's not a good thing. All the little details. But like if you take nor the normal mice and you infect the juveniles, they have a little bit higher airway resistance than the wild types.

But if you deplete the microbiome, the wild type I mean the the mature the older mice still have the same airway resistance, but the juvenile is this much higher. So to me that means the microbiome is having an effect on the juvenile. Sorry.

So they're they're trying to compare the difference between the adults and the juveniles in each case, but what I'm saying is even juvenile to juvenile, there is a difference. So the microbiome is having an effect and it's having more an effect in the young than in the old.

But yeah, I I don't know. I just feel like there's some some things like that that the way they chose to present the data and they're they're making one conclusion for their model, they're ignoring a whole richness of the data, more information that's there. Right. Like the yes, like that's something that Your your the differences are exacerbated when you remove the microbiota, and why is that?

Yeah, and it's true'cause they they tried to s to argue that, you know, if you in in the next figure, they're looking at Muck five A, IL thirty three, TREM twenty Trem two, you know, and they're saying that in each case It's lower in the mature, it's lower in the mature, it's lower in the mature, it's lower in the mature. But if you look at control versus microbiome depleted, it's much higher in the juvenile. So the depleting the microbiome is having a huge effect.

But it's not having an effect in the adults. And I think they're they're ignoring that. Ryom in the juvenile juvenile is actually having a pretty significant effect. Mmh, mmh. So I think if I was the uh head of this lab, I have two projects for my next set of students, one about the microbiome and one about the sex differences. Yes, yes. Absolutely.

Okay, well so definitely more things to explore with the microbiome and the next figure we kind of Okay, so let's let's dive into so what they wanted to then ask was so they're looking at the different cell types that are in the bronchioaviolar lavages of these mice after sendivirus infection um in juvenile and mature mice and They uh

So uh there were differences. Uh I don't you know, so for like for macrophages, there at day eight there was a there was a peak in juveniles, but not in the mature mice. And similar for lymphocytes, although the you know, the the ranges were were pretty wide, but that's something. They've more focused though on the area resistance increase in the juveniles, um, which we already talked about, that kind of decreases by day twelve, so it peaks at day five and decreases by day twelve.

Um, but I think looking at the macrophages, it does this data gives us a little clue into what's coming up next and some of these differences um in the juveniles versus matures. So let's keep we'll keep that in mind. Macrophages. They they next want to look at cytokines. I say, okay, what are the di what are the d different cytokines that uh maybe could define the divergence between mature and juveniles? And they look at bronchial alveolar lavages.

And what they are there there's a whole panel of different cytokines and they're using something called a PCA plot to look at these differences, which is Principle component analysis that basically re reduces the dimensionality of the data so you can easily see like where the clusters are based on the treatment group.

And they observed these different clusters. They observed um ones that were differentially expressed in the juveniles, in the adults, ones that were overexpressed, and they were trying to ask do cytokines correspond with airway disease? And so they what they suggest is that so the the cytokines that are overexpressed in the juvenile mice are positively correlated with disease, but the cytokines that were overexpressed in adults were negatively correlated against disease.

And the ones that were more likely to be overexpressed in the juveniles are things like Ile four, IL five, and Ile thirteen. So it's it's a similar story, I think, to what previous data was showing us is that there's a there's a drive towards TH two based immune responses. And that this can have an effect. post you know, in in the chronic post post acute infection.

A little cytokine data. Okay, so so now what they want to talk about is, you know, it's not just viruses that can potentially induce an lung remodeling. It's also allergens. So they wanted to see if this the same phenotype where you have higher milk five AC and uh higher levels of aeros uh responsiveness and and PAS staining our mucous glycoproteins.

Did that happen in young mice after allergen triggered? And so the allergen they use is alternaria alternata. And I didn't know what that was. So it's um it's a fungus that develops on the fruit. on fruit in cold storage. So would be a a common allergen if you have like fruit a fruit bowl, for example. Fruit in your house. I'm sorry, fruit cold storage, I guess would be in the fridge, but it's common on fruit.

And so after they expose these mice to this allergen, they're looking at ten days post infection. So again, we're not consistently looking at the same time points in these mice in the lungs. And so I I I don't know how these models translate to each other. But what they're summarizing here is that the the there's a different type of response that they still see um induction of mucell metaplasia and type two cytokines in both juveniles and adults.

But there was um less eosinophils, less hyper responsiveness in adults compared to juveniles. And so It these are different responses compared to the antiviral s the the the viral induced responses. And I think that's really all we can say. Um, because they don't take this allergen uh any further. There's not additional experiments. It's really just to suggest that allergens trigger different immune responses in this model of post lung remodeling than viruses. Yeah, I think it's a good thing.

Yeah. Would ideally like to be able to tie it back to that clinical data on allergens um that they had earlier. Um, though we we as we mentioned, um There are some questions on those data. Sure, sure. But yes, I think right. That's a good point. They were trying to tie it back to to clinical data because it isn't just about viruses, but um I g yeah. So just another model of this. Long remodeling. Yeah, and I think that they also, at least at the very beginning, try to s make the comment that

um the allergens are not seasonal in the way that some of the viruses are. Um as as someone who suffers a lot from w all of the trees in spring. I'm not sure I totally agree that allergens aren't seasonal. Um, but I think that's what they're trying to do. Yeah.

What what people refer to as indoor allergens and outdoor allergens, right? So the indoor allergens you typically can't get away from and they're not really cyclical. Whereas the outdoor allergens are cyclical. There's leaf mold in the fall, there's pollen in the spring. Yeah, I know. Might be sensitive too. So this is a very relevant paper for who Brian is what you're saying.

Yeah. So depending on what you're sensitive to. I think it's important though, because if you are a if you're not like allergic to many different things, and we can come back to we were talking about what how you pronounce the word for that. A2PS. Whatever. Um, before we we got on. Like if you're if you're not allergic to a lot of things, if there's a cyclical exposure that every fall you get triggered from leaf mold.

That may be very different than if you're continuously exposed to an allergen in your house, right? because the continuous exposure and remodeling might lead to a different lung pathology or potentially even tolerance. Over time you might outgrow it or something. Um so I think it's it's an interesting thing to think about. Is it the allergen that's different or is it the exposure? seasonality or cyclic or con chronic exposure.

Um so you know the next kind of sets of experiments they do is they they they have uh they do mass cytometry, which um Flow cytometry is how a lot of us typically identify cells, but mass cytometry is is is different in that there you use metal isotopes. to be able to detect different cell types and mass itometry has lower background. Um it can be ideal for image analysis. There's just a lot less background noise and you can potentially h have a lot more markers.

Um and so so they they use mass cytometry and they just wanna they wanna see what are what is different in the immune cells and the lung lavage bronchial alveolar lavages of the juvenile mature mice and and so there I think there's three interesting things happening here. So The B cells are there are similar pr frequencies of B cells in these animals pre infection. They dip in the lungs during infection and then after infection

the B cells in the mature mice are higher. They they go back to baseline, but that's not true in the juvenile mice. they don't really talk about the B cells at all. I think that that is a that's interesting that the the juvenile mice, they do not re the the frequencies of B cells don't rebound.

Um, the other two things that are interesting, and they do comment more on this, I think, as if it's their model, is that the eosinophils substantially, well, I shouldn't say substantially, I mean the percentage of C D forty five positive T cells is like one to three percent, but they increase. over the course of infection which would So lead which would um agree with the phenotype of the animals that they're more TH two bias, leading to increase in eosinophils by day forty nine.

But what they hone in on is alveolar macrophages, and alveolar macrophages are really important um in immune responses in the lung. They can be two types. So there's M1s that are these classic classically activated macrophages, then there's alternatively activated macrophages M1 respond to microbial factors and more teach one pro inflammatory.

Um and then M two are usually induced by T H two and they undergo different types of metabolism and have and and can uh induce collagen deposition and and contribute to maybe Um damaged tissues. So they they see that alveolar macrophages broadly. So they didn't define them here, but they're higher in the juveniles, both. pre and post infection. They dip during infection in both animals, but they really hone in on alveolar macrophages as potentially mediating something here.

Yeah. So so I I wonder about this. So th the way they showed this again is percent of C D forty five positive cells. So if there's a new population of cells that comes into the lung, other things will go proportionally less. And the reason why I think that's important is because you you said there are uh M1 and M2, which there are. There are also two different sources of macrophages. We've got alveolar macrophages and we have interstitial macrophages. And they're actually from different sources.

So interstitial macrophages are developed from monocytes, so they i get into the tissue and they differentiate into macrophages. The alveolar macrophages are embryonically derived. And I always get confused because I thought it was the other way around, but I'm ninety-five percent sure I'm right on that. Um and so those are not the ones that are being recruited.

So how are their numbers changing? And are their numbers changing because they're actually going away and coming back? Or are their numbers changing because lots of PMNs come in and then lots of PMNs go out? Which you see in this data. Like you see n now it's not the same

magnitude in both juveniles and mature, there are a lot more PMNs, which is polymorphic neutrophils that come in in m in the mature, in the older animals. But you're right, when you're looking at the percentage of C D four forty five positive, it's highly sensitive to changes in cells as they move in and out because it's a frequency. So if you have more of one, you have less of another. Right.

Yeah, so if they could show us absolute numbers, um which of course would be challenging here, that would be nice in addition to showing us the percentages. Mm-hmm. Right. Right. So, you know, it took them down a road of of trying to understand maybe the contribution to alveolar macrophages in this hyper responsiveness phenotype, chronic lung remodeling after exposure to to sendivirus, and they do bulk RNA seq and they do single cell RNA seq. And what they pull out is that there's

differences in in MHC class two. And so MHC class two, you know, it's gonna be responsible for processing, I'm sorry, presenting processed antigens. It's it's to C D four T cells and and to stimulate that antigen specific response and obviously critical to uh initiation of yeah, the response after infection. And so they they demonstrate that MHC expression increases with age overall. So it can be induced after viral infection.

But overall it increases. But what was interesting is that at least in the one assay now I don't I don't work in in this field so like you'd have to tell me if you know, they they probably could have done more to demonstrate the antigen processing and Presenting was actually different, but they they'd say that there's no difference in the antigen presentation, even though the expression is higher in in the mature animals.

What do you think, Brianne? Ha ha ha. Uh I think. Look at these bars. 'Cause they they are different actually. They are different and they say there's no difference. Yeah, they even have a statistic there. There's a stat there. I mean there's stats I I I Some issue with it but um but they they say there's no difference and they show there is a difference.

Which would actually I I don't know if it's because it contradicts so the juvenile is in in alveolar macrophages that express what they're saying, lower levels of MHC two. Thank you. they seem to have higher proportion of this this acid uses oval albumin to show presentation of of this processed antigen. And it is higher in the juveniles, which

I don't know. I don't know what you say about that because these are alveolar macrophages that express MHC at lower amounts. Um when you get to the column that says, Okay, alveolar macrophages expressing high amounts of MHC too, maybe maybe they're not different. Um juveniles still seem a little higher. Is this more about the triggerability of those cells? I I I don't know. Right. So I you know, I I think They're transcriptomic data.

I think that that's probably m more clear. I mean it that the they do pathway analysis and they're saying that okay, the the pathways and the genes associated that do seem to be um different and mature versus juvenile. But I don't... really necessarily n know if it contributes to anything other than it's just different in age group. Yeah. I I'm yeah, I'm not sure. I I don't think that this assay that they were using measures antigen presentation.

Because as my understanding is that DQ Ova is highly labeled and is quenched. And so when it's internalized and degraded, it now fluoresces. And what they did was they uh they intranasally injected DQ ova into mice and then they're looking at lung. So This means that whatever the they in instilled was taken up by some cells and

broken, the ova' started to be chewed up, right? In some some compartment where it's, you know, endocytosed. And so presumably those chewed up pieces would eventually go on to an MHC class two. Right, but they're not they didn't measure. OVA getting onto MHC class two. And there are reagents to look at that if they have the right mouse strain, but they didn't look at that. Right. 'Cause you can measure MHC with ovapeptide in it with a specific antibody.

Yeah. Yep. As long as you have the right mouse stream. But they didn't do that. They just looked at did cells take it up and and start to chew up OVA? But most cells will. take it up and eventually start to process it because there's a lot of proteolytic enzymes. So I Yeah. I mean there are Ms. class two positive alveolar macrophages. But I think this just tells us that

at least in the juveniles, the high and lows take it up equivalently and have equal ability to process, you know, to to chew up the ova. Whereas the m more mature ones maybe either take it up less well. or chew it up less well so they have less proteolytic capacity. Which kind of fits with um other studies that have kind of shown that the aged mice have defects. in phagocytosis, in microbial killing in antigen processing, etcetera. Yeah.

think that's a really great point. Um I like how it ties in with what you had mentioned earlier about that question of Are we showing that juveniles are making excess responses or are we showing that mature individuals are making decreased responses? Right. Which one are we considering as the normal? I don't know. Right.

It depends on perspective, right? There's there's something what was it? So when you're looking down, you and the other person is looking down, there's something that you either see one thing or the other thing, depending on which perspective you're looking at it in. There's some kind of meme that I'm I'm not thinking of it, but if you send it you should email it out and then maybe We can figure that out.

So I uh what They're trying to find differences and the and they're suggesting, okay, even looking at single cell transcriptomic data that it's like these alveolar macrophages, they have more KS sixty seven in in the uh juveniles which is proliferation marker and so okay they may be important in juveniles

And me w so what's more interesting I think is that if you were to pull out these alveolar macrophages, so there there are more of them in in juveniles, or at least okay, the percentage is of the frequency of them is different. And if there are a higher proportion of them, maybe that's driving some of this.

You know, are they m two? Uh are they driving T H two? Is that explaining the phenotype? I actually think that there's really interesting things to to look at in regards to alveolar macrophages that could explain this model. But w was not explored. It's like a lot of things left over to look at. Well, I I look at data like these single cell RNA seq and I keep saying this like every paper we look at, it's like they did this experiment to ask one question.

And there is just this incredible amount of data that is associated with that particular type of experiment. that just goes and gets deposited somewhere and people move on to the next thing. So if you're a trainee, if you can figure out how to analyze single cell RNA seq data well. There is just a lifetime supply of data that have already been deposited with new stuff being deposited every day that can be just mined for amazing types of questions that nobody's thinking.

But they're out there. Yeah. And and as you say that and I think compared uh related to a number of other things we've said throughout this paper, um if any of the graduate students current or future uh from this lab are listening to this episode. We're sorry for the to do list that we're we're making. Yeah. You can each have your own project and go start your own lab. There's plenty to do. Plenty to do. Plenty to do.

So, you know, we could talk about the last experiment because they've highlighted these alveolar macrophages as something might be going on in the juvenile. So like let's knock them down in the mature mice. Now, I had to look this up. I've never depleted macrophages from mice. Yes. Uh well, no have I ever no, I've never depleted macrophages from mice. Um max aren't my my main area. So uh they they say that they so Claudrony liposomes? This is what have you used this, Cindy?

Uh, I I didn't do the experiment myself, which is amazing, but but I've read lots about it. And so what happens here is you basically take little liposomes, so that's just little lipid balls. And macrophages love to eat these things. They just gobble them up. And what you do is you load them with a drug that'll kill the macrophage. So the get macrophages that gobble these up, they get killed. And so other cells don't really gobble up these lipids.

So they don't. So there are other phagocytes and they don't like these things? So neutrophils will probably eat them up, but that's They're looking at those. Okay. They're just gonna ignore that. Uh that's I was wondering about this method'cause when I looked up it just seems like a phagocyte. Reduction. It is a it is a phagocyte reduction. However, neutrophils turn over so fast. You're really depleting macrophages and the macrophages don't come back very quickly.

But I mean, neutrophils are w constantly being put out of the bone marrow so they'll just rebound pretty quickly. So if they're in the lung they get depleted, they'll just come right back. But the macrophages, if you deplete them, it takes them a while to come back.

So so I have a question and I will completely admit that this question is based on um the paper that we talked about on TWIV earlier this week that I still am thinking about and I still have a hundred experiments I wanna do and just think it was the best paper ever. or most interesting paper ever. Um, does it does the lipid matter? In terms of which lipid match better. Yeah. I don't know. Lipids matter for mRNA vaccines don't.

Matter for a lot of They do and they cha and they matter in mouse versus human and Ile one in IL one beta and Ile one alpha and Ile one RA production and you should read that paper. Oh. Okay. Or I'll listen to the TWIV and on my drive. Both. Yeah. Yeah, both, yeah. Yeah. I think it's just people say chlodronate liposomes and I think it's just a pretty standard approach that will

theoretically deplete the macrophages. Okay. And the the there's what there's a cool thing that you can do too because I mentioned those interstitial macrophages which are in the tissue in the lung versus alveolar ones which are actually hanging out in the lumen of the lung. So if you instill chlodronate liposomes, it'll deplete the alveolar ones but leave the interstitial ones. If you inject IV,

with chlodronite liposomes, the blood will reach the the interstitial macrophages and you'll deplete them and you can leave the alveolar ones. So you can ac you can do experiments to try and differentiate which cell population is contributing, which macrophage population is contributing to phenotypes that you see in the lung.

Yeah. Do we know I'm looking back in the methods, do we know if they inj in j j infuse them? Them in the nose. So they are depleted in the ICL or macrophages. Yeah. Got it. Um okay, so depleting alveolar macrophages and and after that looking to see If they're not going to be able to do that. our differences and so it seems that the it did deplete them, so that they were successful in doing that. Um and s so the mature animals had higher amounts after claudernate.

liposomal treatment uh of of our so our PAS staining, which stains for the mucoglycoproteins. Um higher levels in the mature animals now compared to juvenile, which it was always flipped before. And this is at okay, the day forty nine and that the airway resistance um after the treatment is significantly higher in the juveniles and they don't take it out to day forty nine. So I'm like, I guess I don't know. It didn't really flip the difference.

Nope. I think what it what they're showing is that in the control liposomes, um so that the macrophages are not depleted, the alveolar max are not depleted, you have a very high airway resistance in the juveniles and a lower resistance in the mature. But if you deplete the macrophages, the juveniles stay the same, but the mature ones come up and look more like the juvenile.

So if you get rid of the macrophages, the mature ones are ha are behaving more like the juveniles. That's what I saw in the airway hyper responsiveness. Mm-hmm. Yeah, similar. Right. Like it it's it's not that the trend flips. But it it's that they come up closer. It brings them closer. Yeah.

So that was figure eleven and you're talking about many figures. That's a lot of that's a lot of data. And I so To summarize, what we saw after COVID nineteen lockdowns was that the amount of kids showing up in the emergency department for asthma was significantly reduced compared to pre COVID lockdown years. Building this mouse model is relevant if we want to try to test some of these hypotheses regarding viral induction triggering.

Acute I'm sorry, uh triggering lung remodeling after I guess forty nine days in this model, depending on the the virus you use, it could be different. But uh in a chronic setting. Whether alveolar macrophages contribute to this, I think is still up in the air. I think they do something. But I don't necessarily think that it was conclusive to show that they they didn't demonstrate that they were the reason why uh, you know, you see these type of type two

responses with higher airway responsiveness in the juveniles. But they do change as the animal gets older. So it's I probably one of the things that contributes. Yeah, I think macrophages are protecting the older individuals from the Yeah. Airway hyper responsiveness. And that in and of itself Then they get more. Yeah. I thought so. Yeah. And why that is, what is it about them? I think more experiments. I think that part of what they're trying to say is that they're functionally different.

Right. They talked about the reprogramming. Right. So something about in the in the older individuals, those macrophages are better at repairing and quieting. Whereas in the younger mice they're more, I guess, inflammatory, or maybe so so it doesn't matter. I don't know, maybe that wasn't the right to say it, but it in the older mice they're more important. The macrophages play a more important role. So if you deplete them, now you're worse.

Right. Right. So that and that in itself is interesting. Yeah. Yeah. Yeah. we can have some data showing how these phenotypes in different age groups are in fact perhaps mechanistically different is a really good thing. Um that it's not just sort of this anecdote of the asthma in the younger individuals and the asthma in the older individuals seem kinda different. Um, but actually starting to move towards what is different about them is an important thing to Yeah.

There's no shortage of uh additional questions to ask. Yeah. Yeah, for sure. Well good. Well, anything else about asthma that we want to say? Any any wrap up comments? So the the the the use of this model is a little weird in my opinion because this is not what happens in people. Right. Not this chronic Lung d you have an acute infection, it comes and goes, and multiple ones of those somehow predisposes. So I would rather they did that kind of model, right? Yeah.

Yeah, an acute infection without this, you know, long term lung injury. I d I just I mean It seems to me that that's not what we know is happening in kids, right? So why would you do that as a model? You might be you're gonna learn different things that may not apply to people. Right. I I think there's advantages to using various different models. I think the disadvantage of this is that you have mice. That's the same thing.

Yeah, I mean they're not perfectly sterile but they're specific pathogen free, right? So they have a pretty naive immune system and you're giving them one insult, waiting and then looking. And like I said, that's not really what happens. We're constantly being bombarded by things that we breathe in, especially when you're young, it's new exposures all the time. We're generating immune responses to it.

And if you're talking about viral infections, respiratory viral infections, you're gonna have one and then you're gonna get mostly better and you're gonna have another one. And then you're gonna get mostly better and you're gonna have another one for your first couple of years, I mean

You you you know this, Steph. I had kissed you. Yeah, the first two, three years of life, you're constantly sick'cause they're constantly sick. Every time they start to get better they come down with something else. And so it's not a one and done. It's a, you know, repeated exposure. And so it's what happens in between those and as you're piling on another one and another one, how is that repair process happening?

And if you're constantly having a high pathology response, you're gonna remodel the lung in a way that's gonna have a long term consequence. And so I think that y Vincent, I do I do hear what you're saying and I I I see what you mean about wanting more acute measurements, but I think to what Cindy's saying, okay, so what we're learning is that

particularly if you have this TH2 predisposition. After a viral infection, you have lung remodeling. So if you're hit again, it's probably going to be worse. And that, you know, they didn't do the second hit, but we could assume if there's more damage that that is what causes piling on of the TH two immune response to cause to cause asthma. I guess I'm assuming there's probably not good like m mouse

asthma attack models that you know that like recapitulate what we see in humans. But uh taking this model and doing a reinfection would be really interesting to try to model that recurrence. Exposure. And also that's not taking into account bacterial infections, right? Because the viral infections predispose to the bacterial infection. You have pneumonia, there's massive lung remodeling. How does that impact all of this?

Yeah. Because the kids that go to the hospital are probably not having a mild case, right? They're probably having a significant event in response to the viral infection and potentially developing pneumonia. Or I I don't know. What is the you know, what is the trajectory versus developing an asthma versus pneumonia? And how do you distinguish that?'Cause you starts off, like you said, with a wheezing, right? There's a wheezing and then a bronchiolitis and then trouble breathing.

And is it just because they do uh an X ray and in one case you see lung involvement and you say yes there's a pneumonia, either viral or bacterial or there's not. Like how do you make those distinctions between the asthma and pneumonia in those children that come into the e emergency department? I don't know. She'll get a clinician on.

Yeah. So we are doing a paper on Twiv tomorrow which is kind of rel related. It's in cell reports. It is asthmatic bronchial smooth muscle increases rhinovirus replication within the bronchi bronchio epithelium. Very relevant. So rhinovirus infection is implicated in the vast majority, the vast majority of severe asthma exacerbations.

And this apparently the susceptibility of the epithelium to rhino infection is increased in people with asthma, so they're trying to get at the mechanism. And what they find is that these asthmatic bronchial smooth muscle cells down regulate the PKR pathway within the epithelium. So Yeah. That is cool. It's not even the target, right? Hm. Yeah. We should coordinate and have like uh themes in Two weeks, yeah. Yeah. Thank you. Yeah. Well, thank you, Steph. Yeah, yeah. Thanks guys. That was fun.

This is really the despite the you know the uh criticisms it's still Makes you think and It's useful. Well, I think yeah, and I think it's good for trainees when we walk through these papers because it's um you know, these are all good things to talk about for experimental design and what would you do next and what would you rather do? And so yeah. Yeah, and I don't think that anything that I I was saying was necessarily criticizing what they showed. It's what they showed and didn't talk about.

Interesting and important. Yeah. It's like, oh look at that significant data but You just ignored it. Or where it was like it's not significant. You're like, what? It is. Right. So I mean I think what we're saying is just that the eleven figures weren't enough. You guys want more experiments and uh I know. Yeah. Well yeah, you wonder could could that have been more than one paper? That's right. Smaller and you had more information about the data that were actually shown.

Right. Yeah. No, I don't know because I can understand why they they Kind of We'll put a little criticism is if this is you who was on this. I think they did a bit of a a a notebook dump. Because I mean you do a giant single cell RNA seq experiment and it doesn't tell you much. Where you gonna put it? You gotta put you in RNA seq experiment, you did, you know, Cytoff and whatever.

It's like huge expensive experiments that showed a little bit. You know, there were a couple of things that you could pull out of it. It was that's not an easy thing to put together. Yeah, no. It's a problem, yeah. People want to publish it. Yeah, it's I can't really. Like even negative data are important to publish so that somebody else doesn't waste all their time doing it, right? Mm-hmm.

Agreed. Yeah. All right, that is immune fifty four. You can find the notes at microbe dot TV slash immune if you have questions or comments. you can send them to immune at microbe dot T V and if you like what we do, consider supporting our efforts you can go over to microbe dot T V slash contribute and uh we are a five oh one C three so your contributions are Federal US tax deductible. Cindy Leifer's at Cornell University. Cindy Leifer on the Twitter. Thanks, Cindy.

Thank you. Steph Langles at Duke University, which is not in Iowa or Ohio. Stephanie Langle on Twitter. Thanks Steph. Yeah, thank you. This is fun. And Brienne Barker's at Drew University BioProf Barker on Twitter. Thanks, Brianne. Thanks, it was great to be here. I'm Vincent Rackaniello. You can find me at Virology. Music on Immune is by Steve Neal. Thanks for listening to Immune, the podcast that's infectious. We'll be back next month.