Scientist Stories: Miriam Merad, The Innate Immune System

This is episode 44 of the Immunology Podcast, the Innate Immune System with Dr. Miriam Merad. Hey everyone, this is Dr. Jason Goldsmith and Dr. Brenda Raud. Welcome back to the Immunology Podcast. We have conversations with immunologists. The Immunology Podcast is brought to you by Stem Cell Technologies., a global biotechnology company supporting life science research and fostering communication and collaboration in science. A quick note about our upcoming schedule. It's our last episode before the holidays, as we will be taking a short and much-needed break. But don't worry, because we'll be back on Tuesday, January 17th, with a brand new episode. Today we have Dr. Miriam Merad from the Icahn School of Medicine at Mount Sinai on the podcast to talk about her research harnessing our understanding of the innate immune system to develop novel therapeutics against cancer and inflammatory diseases. We've also got our usual roundup of recent highlights in immunology news coming up, but first—

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It's that time.

It's the most wonderful time of the year.

Do you know the game Whamageddon? Apparently we're supposed to not listen to hear the song from Wham of some form, and if you do, if you survive it, you go to— you know, you win. It's some Christmas song. It's like an American thing where you try not to listen. The thing is, I always win because I don't know what the song is.

I was about to say, I don't know what you're talking about.

It's some Christmas, really common Christmas song that's on the radio and when you go to the mall all the time. And it's by Wham! And it's a whole game called Whamageddon. And if you lose and hear the song, you go to Whamhalla.

Oh, wow. I only know the Mariah Carey song for Christmas. That's basically it. And that one is really hard to escape, if you want. It is. It is.

That one's impossible. I love the Trans-Siberian Orchestra. I love Carol of the Bells. I'll give that one some props. But that's it.

Yeah, well, but it's still nice, you know. I think December is the best winter month, if you ask me, because January is going to be tough. You know, here in Europe, they almost— they decorate, you know, the streets very nicely, and especially the pedestrian streets are super cute.

The towns do that a little bit here, although I just— I don't know, Christmas starts extending even earlier now, and I'm like, can we have that? Can we just agree it starts after Thanksgiving for the music, please? But yeah, are you doing anything for holidays? Going anywhere? Going to go to like just ski chalet or anything?

Well, I wish so, but no. Well, not too far actually. As this podcast is being released, I'm with my mother in the mountains in the north of Spain, hopefully with some snow.

The north of Spain with snow.

Yeah, of course. Yeah. What did you think? All of Spain is Mallorca?

But of course.

So we'll see. We'll see what winter brings.

I don't get any fun winter plans. Work is a little extra crazy, so I'm just working. But I get to dress up and do my whole LARP Dragon Thrones thing in January, so that'll be good. Okay.

I'm a pirate this time. Then just hold on to the happy thoughts of future reward.

All the happy thoughts of future piracy. All right, well, getting into it, um, check out this segue. I'm going to talk about a cryo-EM structure that's a raft, and pirates use rafts.

There you go.

Flawless.

Flawless.

All right, so the first paper I'll talk about is in Nature. It came out on, uh, 28th of November as an accelerated print. Uh, it is called Cryo-EM Structures of the Active NLRP3 Inflammasome Disk. It's— Li Zhao is the first author and last author is Hao Wu. This is a structure paper. So trying to describe protein structure on an audio podcast is a bit hard. I'm sure on Twitter or elsewhere we will post some pretty cool pictures like we usually do of the papers. But that being said, what I can kind of give at a high level here is they've been trying to understand— so take a step back. Some various complexes are hard to crystallize. Do crystal structures of for obvious reasons, especially the more complex ones that are multi-oligomer or multiple of the same. You can get a crystal structure of 2 or 3 or 4 of the same protein oligomerized together, but when you have heterodimer or heterotrimer or multiple units coming together, it gets harder. And that's one of the cool things about cryo-EM is that instead of requiring a crystal structure, it's perfectly crystallized. So to have protein crystallized, it has to be completely ordered and any disorder is bad news for getting that diffraction pattern. EM is a little more tolerant of that, so it's going to take these proteins and it's okay if each individual protein is disordered, because what it does is it puts the protein on an array and it's going to be at all these different angles. You're going to get all these, uh, X-ray— or not X-ray, but electron shots of it, right? So electron microscopy pictures of it, and then that's computer reassembled into a 3D picture. So you want, you know, 360 views of everything. In each molecule disorder. You just want it pure and have the same thing in each shot, right? So you don't want one protein doing one thing and another doing another. We don't want too many of those because they'll throw them out when you build the model. It's okay if you like 8 conformations, it'll have a problem. But if you can get the one protein to have the same conformation, all of it, you're just going to get a bunch of different pictures. So you don't have to crystallize it, and so you don't have to have that same level of difficulty of generating this crystal structure that can diffract. Then you have to redo it and redope it to solve the phase problem. And so then you have to put in these heavy ions, and that can affect protein expression. You got to do it again. You got to get a second set of shots that look just as good as the first and do the phase subtraction, and it's just hell on wheels. So cryo-EM has really come a long way and been able to solve a lot of structures they haven't been able to do before. It's not as quite good resolution. For example, instead of being 3 or 2 angstrom, this is 5 angstrom resolution, but they are able to crystallized the NLRP inflammasome. So one of our inflammasomes, and they know some really cool stuff about it. It makes this wheel. It's a multimeric protein. In this case, the picture they have has 8 in here, and then they have this ASC adapter as well that has to come on. It kind of like makes a mushroom bottom. So if you kind of imagine NLRP is a, a mushroom top but flat and with the lobes coming on the outside, and there's a bottom adapter as well that comes in. So it's pretty neat to see them build this. And they, they show that you have to have that neck, which is another adapter protein, is not used in the final structure, but the ASC protein is part of the inflammasome concept. It is. And then they show that this region at the C-terminal called LLR interacts with NEC is inhibitory. And so when it's active, it's not there. The other thing they do is they show that you have to have ATP. So then they use ATP gamma-S, which can't be hydrolyzed, and that's required to make the active form. So they're able to actually get an active form of the protein for the first time and then show kind of the important parts of the structure. And then they go in and find some residues they think are going to be important for these interactions. And then they mutate those and show that in that case, in then say cells, uh, that the mutation inhibits the function of the inflammasome, right? So you can show that knocking out it based on the structure gives you the structure-function relationship you expect. And they also then find other residues that are associated with hyperactivation of the inflammasome, where they think that autoactivates without needing the ATP. Those mutations are known in people, so they can now see where those mutations are in people that have this hyperactive inflammasome state. And they can see that that probably, based on what they've seen now, the structure lets it activate without ATP present. And so if you are interested in NLRP biology, and this protein has some very specific unique domains in it compared to some other NLRPs, this is a great paper for really understanding that structure-function relationship. And I think it shows another level of where we're getting with cryo-EM because they had to do some pretty classy protein purification and then, you know, so purify it with a tag and cleave the tag off and do all these things and then mix some stuff together. And they were able to nail all of that. So the EM at the end is probably 30% of all the work that they had to do to get there. And it's 70% is getting the protein purified. But it's pretty cool in terms of trying to see the structure. And they show this FISNA domain is really unique to NLRP3 and it's part of the main conformational change that occurs to make it active. So it's, it's more of a paper to read and look at than just talk about, but it's, it's hot off the press and in Nature because they got a very difficult to get structure. You don't get a lot of structure papers in Nature anymore. And this one you did, you know, the Nature Structural Biology or something, but this is one of those whales that they've been hunting for.

Yeah, I think we've seen a couple of these cryo-EM papers in which they have achieved to really present a very difficult protein and, and, and look into the, the, the, the structure in a way that had never been done before, I think we presented, was it on the beta receptor? How much did we actually know, or was there any type of crystal structure available for NLB3?

I don't think so. I don't think they've ever been. They have inactivated ones where it's just the single protein, but it doesn't make the complex and activate, and they couldn't get that, and they had to add adapters in. They talk about crystal structures that existed ahead of time, but in inactive conformation.

Right. And are there like regions that are harder, like they have more movement or stuff like that and they're harder to characterize or have they been able—

Yes. And so it's easier to characterize in cryo-EM because those will just be blurry and variable sources, portions of the crystal structure if it's floppy, whereas in crystallography, you're just done. You can't get the crystal because it's too floppy. And then the other difference is NMR is a third mechanism. For doing structure, but in, uh, that case it only works with small proteins. If it gets too big, it won't work.

So I think— and NMR is nuclear magnetic resonance, right?

So you typically use it in organic chemistry, but you can do NMR structure and NMR structure dynamics, but the rate limiter for that is like, I think, 40 to 50 kilodaltons, if I remember right, at this point. Quite small. Whereas bigger things are even better for EM. And so a big giant complex of 8 of the same protein, which I don't know how many kilodaltons it is, but even if it was like 20 a pop times 8 is 160. So big complexes they like for, for cryo-EM. So big hard to crystallize complexes or membrane-bound things cuz they can make a membrane disc and then put the, you know, so that's how they're getting these GPCRs that they couldn't do before. And they can also do that with NMR as well. So you can, there's a couple people that can do NMR. On membrane-bound proteins, but those aren't very amiable to crystallography because it's hard to crystallize a lipid. Right.

Yeah, makes sense. Wow. How— well, I guess cryo-EM has been such a revolution in our understanding of small structures, things like that. Super cool. Okay. Talking about revolutions that are, well, not very revolutionary. I'm going to talk about a vaccine that is 100 years old now. That is the BCG vaccine.

The—

what is the name of this? Two French names. The Calmette-Guérin vaccine that has been used for a really, really long time to protect mostly children from severe tuberculosis. But we know that it's not really good at protecting grownups from actual pulmonary disease. Do you, do you, Jason, did you get the BCG vaccination or did your children get BCG vaccination in the US?

Oh no, not at all.

No one ever. That is so weird. Well, that's, you know, I have two BCG vaccinations and you can see the scars on my arm. And that's a very funny thing because you can tell when a person comes from like, I don't want to say third world country because that is an outdated term, but like a developing country because they will, you will find one or two large scars on their mostly left arm, uh, that are because of the BCG vaccination and makes a really big pustule. Uh, and we're still, babies in Argentina are still vaccinated with BCG in their first, I think, 6 weeks of age, but we don't get the, the second, we don't get any second vaccination at the age of 6, which I did get. It's very, this is a very nasty vaccination. So not even you. Interesting. All right. So the, the paper's called Parenteral VCG Vaccine Induces Lung Resident Memory Macrophages and Trained Immunity Via the Gut-Lung Axis. You're going to like this story. Uh, first author Mangalakumari Yeyanathan from the lab of Shaoxing at McMaster University in Canada. It was published in Nature Immunology. So, as I mentioned, BCG is a subcutaneous vaccination that, well, is also very studied. I think especially the beginning of COVID there was this idea that the trained immunity that BCG seems to confer could actually also protect against other respiratory diseases such as COVID. So, there was this idea that maybe people that had been vaccinated with BCG had some kind of advantage against COVID. I don't sure. So, we actually had one of our guests at the podcast, we were discussing this with him. But I'm not sure what is the current understanding of that. In any case, in this paper, they're looking into mouse models. And they want to understand the effect on alveolar macrophages from BCG vaccination. And alveolar macrophages are special cells because those are macrophages that are derived from fetal monocytes, and they populate the alveoli of the lungs shortly after birth. And then they are there, they kind of, they set shop and they persist and they self-renew. So they're not the same as macrophages from monocytes. And What they're— in this study, what they did is basically they looked into how the population of alveolar macrophages is affected and whether they— you can observe the kind of trained immunity. You can see some kind of changes in alveolar macrophages as a consequence of this G vaccination. So they did a— the paper is really extensive. And I have to say, it's a very interesting piece of work. So I'm going to try to keep it kind of high level. What they basically, they do is that, so they, um, they vaccinate mice with VCG, and then after 5 weeks, they look into their alveolar macrophages, and they do see that, um, there are specific changes in alveolar macrophages that suggest an activation. And, um, they express higher levels of MHC class II, TLR2, more IL-6 and TNF-alpha upon activation. So, they seem to be different. These macrophages seem to respond to the BCG vaccination. Interestingly, they also observe metabolic modifications of macrophages, particularly a tendency towards increased glycolysis, which has been associated with kind of activated macrophages and pro-inflammatory macrophages. This effect peaks at 5 weeks after vaccination, and it's really not clear— like, 2 weeks after seems too early. So, this does take a little while to set in. And when they look into peritoneal macrophages, they also see changes there. So, they see that this subcutaneous vaccination has very kind of far-reaching effects on macrophages throughout the mouse. They do also, you know, typical transcriptomic analysis. Again, they see that these macrophages are being activated and show signs of trained immunity. And this is different to what they see if they vaccinate mice with heat-inactivated VCG. So, VCG that doesn't grow, that is basically dead. Or if they have adenoviral kind of nasal vaccination, if you have like a viral vaccination, it's a different thing. When it comes to this alveolar macrophages, they actually— so they show these modifications in their phenotype. But they also become better at, for example, presenting antigen to CD4 T cells. They are better at helping them— they are managing in vitro MTB.

Well, they do—

they infect these macrophages with MTB. And they also see that they are better at controlling the infection. And when it comes to in vivo experiments, uh, mice that are, of course, um, that, that are, uh, vaccinated with BCG, they do better when it comes to my, uh, MTB infection. But particularly, they do better in the early stages, and they have lower CFU in the, in the lung. They are quicker to activate CD4 T cells, and, uh, in, in the— there are mycobacteria-specific in the lung. And interestingly, this advantage that they see seems to actually be largely T cell independent. So, and that's why they really focus on the idea of this— of an increased trained immune— innate trained immunity in the alveolar macrophages. That is independent of recruitment of monocytes from the circulation. Because they show that if monocytes cannot enter the lungs, they still see the advantage. There's something specific about macrophages that are resident in the lung. Lungs. And here's where you're going to like the story because what they eventually show is that BCG basically triggers a colitis, short colitis, that it's like not visible at 2 weeks, peaks at 5 weeks, and it's gone by 8 weeks. And this colitis— yeah, I see, I see your face, your interested face— they triggers a change in the microbiome of the mice. And particularly the metabolome of the guts, the serum, and the lungs of the mice. And so this— they also show that VCG is multiplying, and it's kind of colonizing parts of like the periphery of the gut. And they think that this colonization is what generates the colitis and the changes that they see. So, basically, they end up kind of finding out that these metabolic changes result in certain metabolites, particularly those that are related to the arginine metabolic pathway that end up changing— being reflected in the serum and in the lungs. And this favors epigenetic modifications on the alveolar macrophages that initiates trained immunity. And they can even kind of mimic this effect and mimic this epigenetic remodeling of alveolar macrophages by simply introducing— they have a mix of L-carnitine and butyrate, uh, and they give this in the drinking water of mice, and they can partially re- enact or kind of reflect these changes and this activation, this innate trained immunity of alveolar macrophages in the mice. So, in general, so in conclusion, what they see is they see BCG vaccination goes to the gut. In the gut, it generates dysbiosis, colitis. This generates a change in metabolome. And this all goes all the way to the lungs and modifies the, uh, the status of the alveolar macrophages that are already residing within the lung. And that's how you can— they kind of generate a direct effect on the lung mucosa, uh, from a subcutaneous, uh, vaccination.

And it's permanent, right? Like like this change then persists afterwards?

So the, the trained immunity seems to, yes. Uh, the colitis is, is just, um, they, they, they go back to normal at 8 weeks.

Very interesting. I think that it's interesting we've been using this for how long and we're still learning what it does.

Yeah.

I mean, BCG has been, of course, associated with reduced, uh, disease overall, like like it does, it is known also in humans that it provides protection against unrelated diseases. I think that's super interesting.

It is. It is very interesting and weird.

Yeah.

Good weird.

Not bad. Don't you feel bad you don't have it?

No, because it messes, it causes colitis and I don't need any more of that in my life.

True.

All right. Well, I don't have a segue here other than inflammation immunology. I got no, we're talking Tregs, which stop colitis. So there we go. There's my segue. Um, I'm grasping at straws here. All right, so low-dose IL-2 reduces IL-21 T-cell frequency and induces anti-inflammatory gene expression in type 1 diabetes. First author is Jia Yan Zhang and last author is Ricardo C. Fiera and John A. Todd. It comes out in Nature Communications now, uh, published 28th of November. So this paper is actually a follow-up to a clinical trial where they're looking at low-dose IL-2. The idea of the original trial is that low-dose IL-2 reduces inflammatory states. It's kind of a paradoxical reaction to high-dose IL-2. But if you know anything about signaling, sometimes low-dose signaling and high-dose signaling have opposite effects on receptors. For GPCRs, this is class switching. I don't know the mechanisms besides other ones, but class switching is where a G protein-coupled receptor that's stimulatory or alpha-s flips to inhibitory alpha-i or vice versa. And I think other receptors may have some of this as well, but I actually don't know how they would flip. But it's very common in GPCRs for there to be a dual modality. So that being said, this is all about IL-2. So we're taking low-dose IL-2 for type 1 diabetes. And they basically— this is the follow-up to the FREESIAS trial, um, which is an adaptive single-dose observational study to then really look at the mechanism of action. So the, the high-level version of this is it induces and maintains higher levels of CD4 high, CD127 low, CD25 high T cells and the CD56B NK cells. That being said, it seems they, they fare it down the pathway and then take blood samples and do in vitro work too and really demonstrate that they— this bumps the— this iolodos IL-2 bumps the T-reg component up, um, but doesn't alter natural NK cells at all. And then downstream, they look at single-cell RNA sequencing and understand what happens with the cells and show that it's pumping out general anti-inflammatory cytokines. They look at the frequency of proliferation and see that it does seem to drive some level of proliferation. Then they talk about how previous data with Ki-67 protein doesn't quite match what they see with RNA, that being a proliferation marker. That's because the protein persists a lot longer than a single cell cycle. And so they chalk it up to that, that the clock sync is off between the two of them. And then they say it really, and I don't actually remember what Helios means, but it really, this IL-2 low dose increases these FOXP3 Helios-positive Tregs, and then those in turn suppress IL-21-producing T cells. These T follicular IL-21 cells go down and then there's less inflammatory IL-21. So somehow, and they can't quite figure out, and they look at other cells as well and don't find a huge difference in, they find a little bit of decreased frequency of CD8 mate-like cells, but the dominant signal they can really get from the whole thing is this IL-21. Modality, and it persists. Um, you know, they're doing this 27 days out and it's persisting, and it persists even as far as day 55 into the treatment. So they're really trying to understand the mechanism of action of the existing treatment that they're trying to work on clinically, because, you know, if you're going to develop a drug, you kind of have to have some level mechanism action, and we know what you're targeting, so that when you do your next phases of your trial, you can add this as a biomarker. To know if you have efficacy. And they've really been identified that, you know, this increase in this, you know, the Treg population and the decrease in the IL-21-producing T cells seem to be the big ones. And they, again, they talk about a little bit of innate-like CD8 innate cells going down, um, but they, they don't have as much information on that as the other work. So basically, low-dose IL-2 has the opposite effect. It's anti-inflammatory through inducing Tregs. And so they finally were able to kind of ping the mechanism that it's about Tregs. And then from there, that Treg then drops IL-21.

I mean, I guess that makes a lot of sense. I think low-dose IL-2 has been already for a while known to induce regulatory T cells because they have the CD25 receptor that has a very high affinity for IL-2, so the IL-2 alpha receptor alpha subunit, which is very highly expressed on all types of Tregs, they are really good at picking up IL-2, even at the lower doses.

Right. Whereas IL-2 receptor may not.

Right. Because often IL-2 receptor present in CD8 cells, especially when they're not activated, is less, it has a lower affinity. So you need higher amounts of IL-2 to really activate the CD8 cells. On top of the, of the T-Rex. But I think what's interesting, I think what is not super clear was the IL-21 part.

Yeah, they don't really get at how it's happening, just that it's associated here because it's human data, right? It's hard to do mechanism. Yeah. And it's interesting. So maybe this isn't a case of a receptor switching. It's just an example of preferential receptor binding. And so CD25 has a much lower or tighter affinity. Yeah. IL-2. And so it's selectively at lower doses hitting one versus the other. Or not. Who knows? Maybe, maybe CD25 has a different downstream adapter arms, which I'm sure it does. Right. But like, how is, how is that driving the signaling is an interesting question.

Yeah, I think it's mostly an affinity situation. So that's definitely, definitely Tregs have a very high receptor that has a very high affinity for IL-2. And, and we know that people are trying to like have IL-2s that are either, you know, in a way selective to which IL-2 receptor, because IL-2 receptors have slightly different combinations of subunits. And then they are pressing slightly different, but to some extent overlapping cell subsets. But it is interesting that they did find a little bit more about the mechanism.

Yeah, there's more questions here, but we'll have to jump on. But I do wonder, like, then if you pound Tregs with lots of IL-2, is that deliberately then it goes up even more, or then does it turn off under higher signaling?

I don't think so. So they just take it. They just, they don't downregulate IL-20, if CD25, Tregs.

But do they, does the chronic on state cause an intracellular change in what the cell's doing versus like the lower dose high affinity where it comes on and off? Other receptors will do that where like if you just sit bound downstream on the inside of the cell, it changes what it's doing over time.

I don't, I don't think it's the case of IL-2 receptor. Um, no, no. And it's, and for Tregs, it's super, it's like from the beginning of their, their genesis in the thymus, they depend on IL-25, uh, sorry, IL-2, uh, signaling to differentiate into Tregs. So there's some really cool data. From the lab of Alexander Rudensky, in which they show that early CD25 upregulation due to recognition of self-antigen in the thymus already initiates the regulatory T cell program early on in the thymus. And also, they're looking here, they're also looking into thymus-derived, this HELIOS marker. They use it to differentiate Tregs that are from the thymus. Contrast to potentially peripherally, uh, um, differentiated T-Rex. Uh, so I guess that's why they're using Helios in, in this, in this, um, in their analysis here too.

Interesting.

All right. Okay, but you know what, we're going to keep talking about IL-2 because I also have an IL-2-related, uh, story for you. This paper was published in Science Immunology. So, we have another nice impactful story. IL-2 is inactivated by the acidic pH environment of tumors, enabling engineering of a pH-selected mutain. First authors, Silvia Gaggero and Jonathan Martinez-Fabregas from the labs of collaboration from the labs of Rahul Roychoudhury from Cambridge, Ignacio Moranga from the University of Dundee, and Suman Mitra from the University of Lille in France. And I think this is a very interesting story because it really makes you think sometimes that you— I think it's easy to kind of forget that cytokines, I mean, we add them to our culture, we use them You get very used to just, you know, just shoving them to yourselves. And sometimes we forget the cytokines are biological products that have very specific working conditions, so to say, and then they can be very sensitive to their environment. And this is an example I think is really, really interesting. Basically what they show is that IL-2 and the interaction of IL-2 with CD25, which is the IL-2 receptor alpha subunit, really, uh, is different at lower pHs. And so there is a, uh, kind of the physiological pH around 6.5, 7. And if you have more acidic conditions, IL-2 does not signal so well and does not induce the very important STAT5 activation, which is one of the hallmarks of IL-2 signaling. So, they showed this. They used a lot of in vitro models in which they showed that really there is a very specific downregulation of STAT5 activation when interleukin-2 is binding to its receptor at lower pH. And they do a really nice job at kind of identifying and characterizing the interface and showing that there's some particular residues that at this low pH become protonated, and this disrupts the normal interface between the cytokinin and its receptor. I think they mention a cysteine that kind of seems to be really critical. And this is very interesting because in the case of tumors, when you think about tumors are known— solid tumors are known for generating an acidic environment. They often have very altered metabolism. They often secrete a lot of lactic acid to the environment. And this results in a reduction of the pH. And this can then— you know, you know that IL-2 has issues at lower pH. This might be one of the reasons why T cells have problems with their activation in these environments, because it might be the lower pH that is depleting one of the very important activation signals, which is IL-2. And so what they do is they generate through, uh, directive evolution of— they test a lot of mutation, like mutants, uh, mutant variants of IL-2. And they have an in vitro model in which they can kind of try to, um, Select for variants of IL-2 that actually bind to the receptor at low pH. And so they have a system in which they are, um, they have an IL-2 library, mutant library, and then they are using a receptor tetramer to pick up and identify those mutants that have higher affinity. And they've come up with one particular, which they call Switch2. And they, they show that this Switch2 mutine of IL-2 can bind, uh, to IL, to the IL-2 receptor at low pH. Interestingly, it's kind of a trade-off. It does well at low pH, but it doesn't do very well at higher pH, at like physiological pH. It only works well when the pH is low. So, I think around pH 6 versus pH 7. And so, they show— so, they look a lot into understanding the interface, which are the amino acid residues that are part of this interface and how they are affecting the binding at different pH. So, very interesting for any biochemists out there. Or structural biologists. But what I find very interesting myself is that then they try the switch to IL-2 in a mouse model, a tumor model. And they actually see that it performs a lot better as a— so, in this case, you're using high-dose IL-2 to activate T cells in the tumor. And they actually show that this— it does much better at this. And because it actually doesn't work that well at physiological pH, it seems to have less kind of off-tumor effects because it's not so strong at inducing IL-2 and binding to IL-2 receptors outside of the acidic environment of the tumor, which reduces the off-tumor effects. The kind of the secondary effects of high-dose IL-2, which is nowadays a huge limiting factor for the use of high-dose IL-2 as an anti-tumor therapy. So, you cannot treat patients, get horrible side effects from the treatment. So, in general, so, they do a lot of work in that sense, showing what— that they can actually also in the case of mice models, they can improve the response using this IL-2 with a reduced systemic toxicity upon this therapy. So, I thought it overall was very interesting, this idea of, like, looking closely at your cytokines and what they're doing, considering that we know for a fact that tumors have a more acidic pH, and how they kind of went around this and found an alternative, which might be very promising, uh, as an alternative to traditional IL-2 treatment.

This is interesting.

Modified IL-2, and they think the binding of the, the mutine is perfectly functional at low pH? Yes, they can, they can induce like STAT5 activation.

Yeah, but I wonder what happens at less low pH if it's, if it's, which is the major downstream, um, pathway. From IL-2 receptor. Right. So, so they can mutate it and it works in low pH and it's just fine at more regular pH anyways. You're not screwing it up too much.

I mean, no, it works less. Uh, regular pH.

Which is good since it won't be outside of the tumor, but not off obviously, 'cause then it's not gonna— Huh. It's kinda cool, huh? So, so modifi— modified IL-2. No, that's really neat. Yep. So today's theme is clearly IL-2.

Yeah. Yeah. We finish on a high note. Also, I have to say, I, this completely unrelated, but I wanted to mention, I got help to prepare the papers for today. You know, I actually, did you, did you see all this, uh, OpenAI chatbot that they have been, people have been talking about on Twitter and like all over the place? This ChatGPT thing. So I actually like added. The abstracts and the introduction of the papers, and I asked them to like synthesize the most important points. It was very interesting how it did get it right. Actually did pretty well. It was like as if like an undergrad student would have done it for a second, give it to an undergrad student. Better than I expected. However, however, I also, I got excited. I was like, oh my God, this is going to revolutionize my research. I'm going to be I'm going to be so quick. I want to do these amazing reviews now because it's going to be so easy. So I actually asked the, the, the, the bot to get some clinical trials on some of the topics I'm interested. And you, you know what it did? It made up clinical trials. Like it gave me names of clinical trials with clinicaltrial.gov, like numbers, but those numbers did not belong to any trial. If you actually looked them in the website from clinicaltrials.gov.

Just made some up for you. Uh-oh. Yeah. Hasn't had academic integrity yet.

Yeah. What a sneaky little thing. So I was a little bit disappointed. I thought this was going to be— I was going to start doing half of my job, but apparently not. But it did help, you know, synthesizing the papers for me.

So that's a start. Crazy. I got nothing there. All right, I don't even have a segue. The robots are to take over the world. But before the robots take over the world, we are going to be speaking with Dr. Miriam Merham at the Icahn School of Medicine at Mount Sinai in just a moment. But of course, before we get to that, we want to remind you that Stem Cell Technologies has amazing wall charts for you to hang up on your lab. You can get all types of a variety of interesting immunology topics, including a snapshot of COVID-19, everyone's favorite pathogen, an overview of antigen processing and presentation, and much more. Explore all of the Immunology Wall Charts and order your free copy at stemcell.com/immunologywallchart.

Hi everyone. Joining us today in the podcast is Dr. Miriam Merad. She is the director of Precision Immunology Institute at Mount Sinai School of Medicine in New York. And I'm very excited to talk to her because Dr. Merad's work has been instrumental in our understanding, particularly of tissue-resident dendritic cells and macrophages. Their origins. So I'm very happy that she's with us today. So welcome to the Immunology Podcast.

Thank you.

Thank you for having me. Thank you for being here. All right, Brenda, you got the enthused smile going, so why don't you go first?

Why don't we start a little bit back in time? Because I think it's fascinating. Your lab was the first one to identify, amongst others, so amongst other contributions, scientific contributions, to identify tissue-resident macrophages as opposed to monocyte-derived macrophages. And I think that nowadays we kind of take that for granted. We just discussed in the paper roundup alveolar macrophages, for example. But we didn't always know this, this idea that some of the macrophages just come there, are there from the beginning, and they're just resident in the tissue. Similarly to other cells. So, I would like to maybe start for those of us who are just not very knowledgeable about what is our current understanding of where macrophages come from, what are they doing, and maybe also a little bit about dendritic cells, because it can be so complicated to keep track of all the subsets and their origins.

I will tell you one day about the history of this beautiful story of of how scientific inquiry can really transform dogma, right? So the dogma at that time was that macrophage and dendritic cells, when I joined the field, which was I think in 1998, right? This is where I started my PhD, in fact, Stanford, after finishing residency in medical oncology in Paris. The dogma at that time was that these dendritic cells and macrophages, which I was very interested in because of their role in antigen presentation, these cells came or derived from circulating monocytes. And that was that, right? And but there was very, very little data that suggested that was the case. So now fast forward 20, almost 20 years later, really the understanding of the ontogeny and origin of the cells had significantly expanded and changed. So it's first in terms of dendritic cells. In fact, dendritic cells are not as complex as we think. And I'm going to summarize them here for you. There is two subsets that we should care about, a subset that's called DC1 and DC2. These two subsets are present in all tissue, non-lymphoid tissue and lymphoid organs. They arise from DC-restricted precursors that in the bone marrow, and these DC-restricted precursors come from myeloid progenitor. And the engagement into these DC-restricted lineage is dependent on a growth factor, an important growth factor called FLT3 ligand that is produced in the bone marrow. And really pushing that FLT3 signaling promotes myeloid engagement in the DC lineage and commitment to that lineage. You know, this is how you progress in hematopoiesis. You reduce your ability to differentiate into one lineage, acquire the ability to differentiate into another lineage. And this is really dictated by the production of cytokine and the expression of cytokine receptor. It's unclear how you acquire those cytokine receptor and ability to respond to ligand. And there is potentially a stochastic effect and some specific niche in the bone marrow. This clinical research, which is ongoing. But this is what's happening for the DC lineage. These DC-restricted precursors start to differentiate into DC1 or pre-DC1 and pre-DC2 in the bone marrow. They circulate in the blood in their pre-DC1 and DC2 stage. They arrive in the tissue and they differentiate even during homeostasis into DC1 and DC2. This is homeostasis. In an inflamed setting, there is now a lot of inflammation and that differentiation into In fact, myeloid cells is a little bit complex. So myeloid progenitors, monocyte and dendritic cell-restricted precursors, all can differentiate into a dendritic cell-like cells in this inflammatory setting. And the cells that we now have in addition to the homeostatic one that I just described are called DC3. And these are like DC that differentiate into in this inflammatory setting. When you look at the molecular program of DC1, DC2, and DC3, They are very distinct. And this is why I think we should understand their contribution, the different contribution of these compartments separately and into any type of outcome, disease outcome. So that's the dendritic cell. So it's quite simple. And the reason why sometimes it's complex is because we somehow mix a molecular state of a DC with a subset. A subset is a cell that has a specific lineage origin, right?

They come—

so, you know, I'm a hematologist by training, by clinical training, and an oncologist. So I studied hematopoiesis quite a bit. And it's been extremely useful to integrate— and this is what I meant about the history of macrophage— integrate that hematopoiesis background in immunology. Right. And it's really that at the interface of these two fields that I have made, you know, these discoveries, this understanding, this realization that you understanding what the cells come from matters in terms of functionality. So it's very important to know that a subset refer to a cell that have a specific origin, that subset can acquire many different molecular states, right, in response to different types of injury. But those molecular states should not be called subsets. And sometimes this is a mistake that is being made in the literature. And therefore, you know, it may appear more complex than it really is, right. And the way we all define, you know, these different subsets by using fate mapping models of progenitors so that we know what we are talking about. And this is how we know that it's the molecular state of that particular subset because of this fate mapping strategy. Now, we have used exactly the same type of strategy to look at macrophages. So macrophage is even more interesting because macrophage, we have talked to all macrophage, in fact, ontogeny work has been done in the context of inflammation. Right. And before understanding inflammation, that's what I tell immunologists all the time, is that study homeostasis because then it's much easier to understand inflammation. If you know exactly how the tissue should look like in terms of cellular composition, molecular composition, cellular interaction, when things don't go well, you know, you know, you know what's pathogenic and what's homeostatic, right? So in macrophages, you know, all the studies that I was looking at, so I was quite interested in understanding how these cells, you know, as the hematologist, as the cells differentiate, where are they localized, how they interact with T cells, because I was interested in their antigen representing role in the context of cancer. I was in fact sent to do a PhD on DC vaccine. I did something very different. And so I was very interested in understanding the first origin of macrophage before understanding how they contribute to tumor immunity. And what I realized at that time, so I was at Stanford, that there was really very little data on homeostasis. All the data that established the role, the contribution of monocytes to macrophages was done in the context of inflammatory setting. So, using a lot of gene mapping models, what we realize, in fact, initially parabiosis model. This is a model where mice are attached at the joint and at the— well, they are attached surgically. It's a little bit cruel, but we try to minimize the pain that is associated with this. With the surgery and with the experimentation. So these parabiotic mice, when you attach them, they start to share the same blood circulation. And if we now use mice that have different tracer in the hematopoietic compartment, for example, congenital mice that express different allele of CD45, you can then distinguish blood cells from one parabiont and the other. And this is a way for to that enables the tracing of the physiological turnover of hematopoietic, physiological differentiation of circulating precursors to, let's say, immune cells in tissue.

So you talked a lot about DCs, but for macrophages, I think I want to make it really clear that we realize that they come from multiple places now. And I think that's one of your very first discoveries that, you know, kind of put you on the scene. Can you talk a little bit about what we now understand, because we used to think that all macrophages just came from the bone marrow and floated through, and that's not true.

And so what is true?

Right.

So, so now the current understanding is that monocytes, macrophages come to bone marrow only during inflammation. This is where monocytes are released in excess from the bone marrow, come to the tissue, differentiate into what we now define as monocyte-derived macrophages.. In the steady state, all tissues are populated by macrophages that arise from precursors that originate during the embryonic setting, infiltrate the tissue during embryogenesis, and then stay there for a very prolonged period of time and contribute to this population of tissue-resident macrophages. So, these tissue-resident macrophages have a very important role in the maintenance of tissue integrity, in addition to their immune function role. Whereas the monocyte-derived macrophages, we believe, have mostly an immune role. They come here to help clear a threat. And this is their main function. And this is why it's going to be very important to understand the contribution of these two compartments to disease outcome, the outcome of any injury. And this is a big focus of my lab right now.

And these resident macrophages, they also generate niches and they renew themselves. They can maintain their numbers throughout the life, the whole life of the individual.

No, absolutely. This is what's very interesting. And many of us are still trying to understand how they do that. But you mentioned niche, and that's very important because they are, and I think this is what's very important to understand, because they populate the tissue very early during development, they occupy these very specific niche and they are imprinted by that microenvironment. And they're imprinted so that they serve a function, right, that has been really shaped by needs, right? And understanding that, I think, is going to really continue to lead to many discovery in terms of tissue homeostasis, tissue repair, tissue regeneration. There is still much to discover.

So this may not be known yet, but have we been able to experimentally in a mouse or whatever, deplete all of the tissue-resident macrophages in a single tissue and then see how it restores? Have we figured that out yet? Or if it can, or do you have to have some 1% that survive whatever you're doing to restore them back?

That's a good question. So, you know, we've done that quite a bit. In fact, it was a big focus of my lab for many years when we started. You can deplete them and in fact, they repopulate themselves, but you never deplete them completely. So, the belief now is that they repopulate. The few ones that remain are repopulated. The exact mechanism of repopulation is still being understood. We still have to, I think, continue to build knowledge there.

So, within this category, we have a lot of cells. So, we have alveolar macrophages. We got microglia as well would be in this category. Langerhans cells, all these cells that we study maybe in our general immunology, they're all part of this special group of cells. Maybe we can talk about a specific case because your lab, in a more recent work, you actually study, for example, lung macrophages in the context of COVID-19. Maybe how— could you maybe talk us through it a little bit through this contribution and in the lens of our understanding of, for instance, in this case, I would assume lung macrophages or alveolar macrophages, how do they help against the disease? Or maybe even more importantly, to prevent the excessive damage that is characteristic of COVID Yeah.

Well, that was a fascinating study, I have to say. Difficult one, because it was during the pandemic, but very interesting. So what we realized is that patients were dying of inflammation, all of us saw that quite early. And there was this this category of patients that were very susceptible, right, to this disease. Patients who in fact have some type of vascular damage or chronic inflammation, you know, including patients with diabetes, patients with hypertension, age. Age was really the biggest risk factor for severe COVID disease and obesity. So there was a group of— and really infant who usually are also quite susceptible to novel viral infections, were not really at risk for severe COVID-19. So very quickly we started to think that there is a repair issue. There is an inability to really repair damage that is probably stronger in infants. There may be other reasons for that, but that was our hypothesis when we started this work. And what we— and maybe in those patients that older patients and patients with this chronic inflammation, something that was wrong in their tissue already. And what we find in this study is that indeed we believe that a damaged alveolar macrophage pool, which is the tissue-resident macrophages, which are the first cells that also can sense damage, right? They sense, but the way they sense and they call for help. So they induce that first inflammatory response to bring other immune cells to come and help clear the damage. That type of response is going to set the tone for any type of inflammatory lesion that you are going to have. Right. So if you do not have that first alert, right, the virus can propagate a little bit more than it should be and maybe creating more inflammation and more damage. So that may have been the case. But the second thing that alveolar macrophages do, so they call for help very early. Right? So this is why you can contain the threat faster if you have a strong tissue-resident macrophage pool. The second thing that they do is they also modulate inflammation. They know how to modulate it. They can respond to toll signal, to microbial signal, but they know also how to contain inflammation. So the risk that you have when you don't have a strong macrophage, tissue-resident macrophage pools, we believe is that you have excessive inflammation. And this is something we are starting to be very interested in, especially in the context of aging, where we think that these tissue-resident macrophage pools across different tissues is probably altered. And we are trying to understand whether this alteration is potentially contributing to chronic inflammation that we see in older patients.

So you think this aging question in aging tissue-resident macrophages is partially due to the fact that these cells can age. So what I mean by this is obviously the bone marrow ages too, but it's a continually renewed pool, right? So at baseline, yeah, the stem cells in your marrow are going to age and so your cells will be a little older, but you get new macrophages and new blood cells all the time. But the tissue-resident ones are only going to change over when something bad happens. And so they're going to be subjected to kind of long-term senescent aging in a way that a bone marrow-derived macrophage won't? Do you think that's part of this unique physiology here, that they age kind of like our muscles and, you know, our organs do in a way?

Yeah.

So it's possible. It's not— it has not been studied. And so one of the questions that we have is how they age indeed. And, and using this gene tracer that I introduced earlier, we are looking at these two compartments of of monocyte-derived macrophages and tissue-resident macrophages and whether they age differently. Because our hematopoietic stem cells also age. So, it is possible that that monocyte-derived macrophage compartment is also altered. But however, you know, the one thing that we are exploring is that these tissue-resident macrophages are in these niches that we discussed. And one of their roles is to constantly clear damaged cells. Right? For microglia, for example, it's very clear that their role Clearing these damaged neurons is key. Right? There is, you know, this fantastic data in the Alzheimer's field that most of the susceptible genes for Alzheimer's disease are in fact the genes that reside in this macrophage compartment. Genes that regulate, in fact, macrophage function. So, a disruptive macrophage function is significantly associated with a risk, an increased risk of Alzheimer's disease. So our belief is this is true everywhere, is that somehow the ability to clear these damaged cells, which is part of homeostasis of any type of tissue, is very important. And this is likely disrupted during aging.

I have just to touch on one more area of research of your lab in the context. I think you mentioned also we're talking of lung. So, we know— so, now we talked about macrophages and resident macrophages in the context of infection. What about the kind of the opposite situation in context of more of cancer? And we know that it is a little bit— macrophages have been associated with worse prognosis, best— better prognosis. Mostly, I think mostly worse prognosis, those anti-inflammatory macrophages. So what is our, maybe in a kind of a high view, what is our understanding of the role of these resident cells in cancer?

Yeah.

Okay.

So that's a very important question. Right. And this is a big focus of my lab because I am a hematologist oncologist by training. So this is, this is why I went to research to understand how we can harness that immune microenvironment to treat cancer. Big focus is these macrophages. And I think it's going to be, and there is still a lot, a lot to discover.. And, and we need to think about macrophage, in fact, in a dynamic manner, because there's one finding that we had and we published recently is that the tissue-resident macrophages are the first to interact with cancer cells because they populate the tissue. So when the cells have become malignant, they start to produce very quickly some inflammatory molecule. And you see these tissue-resident macrophages going close to cancer. We've in fact followed using again the gene tracing strategy that we've been using for years, macrophage interaction with cancer during tumor progression. So, and they also interact with the stroma. So these macrophages are going to build a niche for the tumor to progress. At some point during tumor progression, these T-shoid macrophages are excluded from the tumor lesions and there is a big recruitment of monocyte-derived macrophages. So we are starting to understand the contribution of this compartment, contribution to early invasiveness, contribution to progression, contribution to metastasis. And now we are just starting some work with a fantastic cancer biologist, who is looking also at dormancy. You know, cells, it's micrometastasis that take places in a tissue and what he sees in collaboration with my group and with tools that we generated is that the dormant cells are always surrounded by macrophages. So it's possible that macrophages decide whether a metastasis is going to be quiet and never reactivated, or that, you know, that the tumor cells is going to grow. There is so much to still uncover, and I'm encouraging those interested in the subject at least in cancer immunology, to really focus on, on macrophages.

This is where the next set of big targets against cancer are going to come from. So to kind of shift gears a little bit and talk more about scientific work broadly, so maybe the segue is big targets, big things. You're also the president of the International Union of Immunological Sciences. Can you tell us a little bit about that union, especially for our American audience?

Who may not know of it. Yes. So I just became the president of the Immunology Society, International Immunology Society, a week ago. And, and it's a big— it's a big job. There's 87 societies that, that are part of this union, 60,000 members. And, and really one of the goals is to share knowledge between all these different societies. Exposed, less exposed. But one thing that I've said in my address recently to all the presidents of the different societies is that, so we have, you know, the Western world, you know, societies that are very active, you know, quite rich. Now there is clear path in the clinic. So we are booming, right? And there is the developing world, which are, you know, less able to do this type of inquiries and scientific endeavor that we do.. And yes, we have to expose them, but also selfishly, we can learn from this country so much, right? Because they are more exposed to infectious disease or infectious cancer. And therefore, even if we are not interested in scientific sharing, and we should really engage so that we learn from all these different pathologies that are still endemic in many of these countries. So I'm hoping that what we'll be doing is really trying to build endeavor across different countries that will continue to help us understand the regulation of the human immune system. You know, in Africa, for example, there are patients that have these chronic parasitic diseases, but they live well. They don't— they have no symptoms. And you may have a patient in the Western world with the same infection and could die from it or have a very severe inflammatory disease. So how do immune systems cope with these chronic infections? A big question, right? And the answer are in some of these patients that live in these countries.

So I'm looking forward to interacting with many of my colleagues in the developing world. So you are— the union is now meeting, right? In 2023, you are actually going all the way to to South Africa for your— for the Congress. And I think that's probably a nice effort to bring the international immunology to, I would say, the Global South a little bit. I come from Argentina, so sometimes it's hard, especially immunology being such a complex, expensive, if I would say, discipline to research. I think it's important to try to bring it more in a more kind of equal way, right?

Absolutely. So this immunology congress is the largest immunology event. So it happens every 3 years and has never been held in Africa, right? So it will be the first time that this meeting will gather, right, in Africa. And it's the first time that we meet after COVID also. So it's a big statement for us. Us, right? And yes, it's expensive to go to South Africa, but it has been extremely expensive for African trainees and faculty to come to our meeting also. So it's very important that really we all take up the challenge and go there and meet our colleagues and discuss about global science and closing the healthcare gap, you know, between all— between continents, right? We have so much to discuss, and I'm I'm so happy that we are meeting in Africa. The challenge is that I'm hoping that people will come, right, and will subsidize their trainees to come because we want— faculty will come, they're very excited about South Africa. We want the trainees, the American trainees, the European trainees to go to Africa and meet with African trainees. So we are going to try to fundraise for African trainees first and also for European and American and South American and Asian also to go there. But I'm very excited about the meetings. We will talk science, but we will also talk policies. We will also talk about young immunologists. We need to think about how we can also change the way this career of ours, which is too long and too tedious, is developing. And I'm hoping that there will be a lot of young immunologists. In fact, we will have several debates and we hope to hear from young immunologists about the way— what they think about this career in basic science or in translational science or as a physician scientist. How can we shape it differently, right? And so there will be many opportunities to talk about. We will talk about publishing, the future of publishing. We will talk about misinformation. We will talk— we will have debates on also very interesting questions, tissue-resident immune cells versus circulating immune cells beyond macrophages.

It's going to be a super fun meeting. So can you remind our listeners, when is the meeting planned for?

Yes. So the meeting is November 27th to December 2nd, 2023, in Cape Town. We have a fantastic lineup of speakers, several Nobel laureates, faculty that are super engaging. We will have speakers from all over the world, including from Argentina, several speakers also from Africa that are joining us. We will have the Minister of Health of Africa talking to— of South Africa talking to us also. Many editors will be there.

I'm hoping that you guys will also join us and do a series of podcasts there. I'm renewing. I'm getting my passports renewed for all types of stuff early next year. It takes forever in the US to do it, so I have to start. I have my passport always ready. Well, you can't, but they They take it from you when you renew it in the US. So to renew it, you have to turn it in. Really?

I pick a period of time I can't travel.

Oh, so please renew it as soon as possible. Sounds very exciting. There's so much to learn from everywhere in the world, which brings me to our last question of the day. Uh, and I think I might have an idea of what your response would be. If you were not a very successful, inspiring scientist as you are, what do you think you would have done with your life?

Well, before going science, I went, I went to medicine, but I hesitated. Well, I hesitated between different things, but you know, I come from Algeria. We didn't say it usually. I'm always asked where I'm from. So Algeria, North Africa, and I left my country when I was 18 because of many political troubles there. So I was raised in a place where I wished governments were thinking differently about a different type of policies. And so I hesitated between joining, you know, thinking about political science. I suppose I didn't do it because I wasn't sure whether political science was rigorous enough. You know, I wanted to have to understand the technicality of it. But I am interested in, I would've been interested in thinking globally and strategically of how we lead a country, right? And I'm hoping that this is what I will do in my new role as a president is be strategic. You know, sometimes what we forget, right, when we become scientists is that we are consumed by the process. I need to write a grant, I need to write a paper, and we forget what the mission is, right? The mission is transform medicine, no matter of, at least for immunologists. And in the country, it's the same thing, that politicians forget that the mission is to improve the life of their citizens. It's not the process of being a politician. So I suppose I have used the same line of thought in the way I approach medicine, is have an impact. We need to have an impact. It's much more fun, in fact, to have an impact in life than to think of how I can survive in a job of mine, right? And I'm encouraging, in fact, and my students in the institute that I lead at Tsannai know that I'm encouraging scientists to go into politics because we always use, you know, we have this data-driven mind, right? The rationale, we use a rationale for any decision that we take. So we have the perfect training to in fact contribute to society. In fact, we should remove this word politician. We need to contribute. We servant. We are civil servants and we need to contribute to improving our society.

And I think scientists will do that very well. I have to agree with you. That's our—

that's a great way of putting it. Well, we had a German chancellor who was a scientist, but I was going to point out that you live in what's almost a small country. Any chance you're going to run for mayor of New York then?

No, no, because I became addicted to science. And also I have, I think I need to, I will have to improve my accent a bit more before running.

Fair, fair.

New York, they will judge you that way. Well, as long as you don't sound like you're from New Jersey, I think you'll be fine. Sorry, New Jersey. I don't even know what I'm talking about. I've never lived there.

That's how I hear it goes. That is a true stereotype set. All right, like all things good, things that must unfortunately end on the podcast, but thank you for coming on. Thank you for having me. That brings us to the end of our show. Don't forget to subscribe to our newsletter at www.immunologypodcast.com to get the show notes, including an episode summary, links to all the interview and roundup papers. You can also reach out to us on Twitter at @Immunopodcast or by email at info@immunologypodcast.com. With feedback or to suggest guests.

See you next time.