Okay, great, so welcome everyone for today's session about it's not just macrophages anymore and nuclear receptors. My name is Laura Solt. It's not just macrophages, but let's not discount them, they're still really important. Very true. My name is Laura Solt. I'm one of the chairs today, and my co-chair is... I'm Eric Nelson from the University of Illinois. So welcome. And our speaker this morning is going to be Ines, sorry, I apologize if I butchered your name, Ines Bineta-Torre from University College London. She's going to be telling us about LXRs and CD4 T-cells and metabolic control. Okay, so let's start. So thanks, the organizers, for giving me the opportunity to present the work of my group with you today. And the overall goal of my group has been to understand the crosstalk between lipid metabolism and immune responses and how these pathways affect the progression of diseases, mostly cardiometabolic diseases, but also autoimmunity. And we focused on the liver X receptors or LXRs and also the transcription factors like the interferon response factor, IRFA, but I'm not going to talk about that today. So the LXRs have already been introduced in other sessions by Laszlo and Eric. These are lipid-activated receptors that partner with RXR, and we use a few synthetic molecules like the GW3965 as the LXR agonist and G233 as an LXR antagonist. And these receptors are really considered master integrators in lipid metabolism and immunity. Sorry. Mostly, they were initially described, a lot of their biology was described in metabolic tissues like liver, but also, as we say, macrophages. And in terms of lipid metabolic pathways, they have been associated with different level of excretion or synthesis or export of different lipid species like cholesterol, fatty acids, triglycerides, and phospholipids. So our previous work focused on the role of these receptors and mainly LXR phosphorylation or LXR-alpha phosphorylation in particularly in the regulation of whole body or tissue lipid homeostasis and the progression of cardiometabolic diseases. But we changed gears a few years back, and the work that I'm going to present to you today is focused on the regulation of membrane lipid metabolism. And this is the work of an excellent PhD student at the time, Kirstie Waddington, and the result of a wonderful collaboration with Liz Jury, who is a true immunologist and T-cell expert at UCL Division of Medicine. And I will obviously, because of this session, I will be mainly showing our work on T-cells. So we've been studying T-cells for many reasons. They're very important cells for recognizing antigen-presenting cells, and the lipids in the membrane are essential and play an essential role in the formation of the immune synapse with antigen-presenting cells. So upon the recognition of the T-cell receptor with antigen-peptide-bound MHC2 complexes in the antigen-presenting cells, there's an assembly of a large signaling complex that triggers a signaling cascade, and this promotes different effector functions, proliferation, and cytokine production. And we normally tend to depict the plasma membrane as a simple, smooth lipid bilayer, but the reality is that this is a much more complex structure with lipids and proteins, but also carbohydrates, and it's the interaction between all these components that actually promote protein dynamics and impact on cell function. So the lipids in the plasma membrane are also quite heterogeneous, and they're organized into micro-domains that we call lipid rafts, and these rafts are in what we call ordered phases as opposed to non-ordered or disordered phases, and they're enriched in cholesterol and glycosyl lipids. And these are the areas in the membrane where the receptors, the membrane receptors, tend to localize, like the TCR or T-cell receptor. So the cholesterol is going to maintain the structure of the lipid rafts, inhibit a spontaneous TCR activation and promote the clustering of these receptors, and it also promotes and regulates proliferation and differentiation of these cells and cytokine production. And the glycosyl lipids are also going to impact TCR membrane signaling and responses to stimulation with cytokines and T-cell differentiation. So from a biochemical point of view, these lipid rafts really help coordinate the protein interactions in both resting and activated cells. And in the past, these structures have been a little bit controversial, but now we have the tools to visualize the lipids in these membranes and in these rafts, so we use Philippine binding to measure membrane cholesterol and also cholera toxin B to measure glycosyl lipids in these lipid rafts. And what we knew when we started these studies in terms of what was known on LXR activation and LXR biology in human immune cells, mostly, again, was focused on monocyte-derived macrophages of differentiated cells, and there were both pro- and anti-inflammatory roles that had been described for LXR. And in terms of T-cell biology, there have been studies linking the LXRs with regulation of proliferation, homeostasis, and also things like infection and responses of some T-cell subsets like Th17 and Th1. So to define better the transcriptional actions of these receptors, we performed global transcriptional profiles in cells that were exposed with the LXR ligand, so specifically CD4-positive T-cells. And as you can see here in this volcano plot, there were a number of changes of LXR targets that were regulated. Some of these targets that we identified were already well-characterized targets for LXRs in other cells like the macrophages, such as the ABC cholesterol transporters, but also the SIRP1C fatty acid synthesis transcription factor, and we also identified novel targets such as the long non-coding RNA, Olmalink. And perhaps not surprisingly, a lot of the pathways in reaching this data set had to do with lipid metabolism, various species of lipids such as cholesterol fatty acids and phospholipids. And we wanted to explore this further, and we performed lipidomic analysis in these cells that were exposed with LXR ligands. And as you can see here, the total lipids were not affected, or the total lipids, as you say, that are measured by this platform were not changed when the cells were exposed to the LXR ligands. But we could see changes in about 15% of the lipids we explored. And among these, there were the triglycerides, but also exocytoceramides, which are modified forms of ceramides, and you can see the quantification of these changes here in this graph. So we saw a true shift from ceramides to exocytoceramides, and this shift can be promoted by two different enzymes, UGCG or UGC8, but we only saw the expression of UGCG in these T-cells. And actually, the increase in exocytoceramides, because exocytoceramides are precursors for glycosphingolipids, so we thought that this increase could be translated also in increased levels of glycosphingolipids, and this is what we showed here, the membrane glycosphingolipids were increased in the cells exposed to LXR. Again, this is associated with increased levels of UGCG that were confirmed in many different independent donors, so every dot you see there in that graph is an independent donor. And indeed, we were able to find an LXR binding site in the UGCG gene, so this is a true novel LXR target. I've mentioned that we saw changes in ABCA1 and ABCG1 cholesterol influx genes, so these changes were pretty massive and to a level and to a degree that we haven't seen in other cells like the macrophages, so we wondered whether these would be translated into cholesterol influx and also different levels of cholesterol in the membranes of these cells. And this is what I'm showing here, so the cholesterol levels in the membranes of cells exposed to the GW ligand were reduced, and the overall glycosphingolipid cholesterol ratio in these cells was induced, and this is in resting T cells. So I remind you that cholesterol and glycosphingolipids are going to influence the lipid membrane order, and this is important for the interaction of the membrane receptors and can alter the strength and the nature of the signaling events and impact T cell function. So we use a fluorescent probe to measure lipid membrane order, which we call for short A&E, and this is a fluorescent probe that signals differently depending on whether it sits in areas of high membrane order or low membrane order, and as you can see here, the cells that were exposed to the LXR ligand show a decreased membrane order, and this is quantified here. This is by confocal microscopy, but we were also able to confirm this by flow cytometry, and you can see this in the blue bar here, and this effect was reversed when the cells were exposed to the LXR agonist GSK233. And in fact, we were also able to see that certain target genes of LXR are preferentially expressed in cells that overall show a low lipid order in their membranes, as you can see here for UGCG or the cholesterol transporters, ABCD1 and ABCD1. So the link between LXRs and membrane order is not new, and it had already been described in mouse macrophages, but it was through a different mechanism, so this is the work of Peter Tontonos, and he had shown that LXRs were regulated membrane order mainly through changes in the phospholipid composition of the membranes to actions regulating the enzyme LPGAT3, and also at the level of cholesterol regulation through changes in ABCD1 transporters. But interestingly, it didn't seem that glycosphingolipids were affected in these cells, and indeed when we looked at a comparison between our T cell datasets of target genes and what was described in the literature, and our own mouse macrophage datasets as well, it seems that there's only a partial overlap of target genes, and there's a subset of targets that are preferentially regulated in the T cells by LXR-alpha, and actually UGCG is one of those genes. So what I've shown was in resting cells, so how about activated T cells? So as you can see here, we see pretty much the same thing. The glycosphingolipids are increased by GW, and the LXR agonist and cholesterol is reduced, and this is also translated into changes in mRNA expression. I don't have time to go into all the details of this, but basically there's some target genes that are going to be differentially regulated by the LXR ligand, either in cells that are activated or when the TCR is engaged, or when it's not engaged, they're going to have a different regulation, and for example, I'm showing you here that certain oxysterol metabolism enzymes, including CYP27A1, are reduced or are regulated when the cells are activated by the TCR, but we're still trying to figure out what these changes in oxysterol metabolism enzymes mean in terms of T cell function. So do these changes that I've shown you in the membrane lipids and membrane order impact the function of these cells? So one of the first things that we studied was the immune synapse formation and membrane order at the level of the immune synapse. So this is what you can see here. So initially when the cells are activated, you can see that there's a reduced membrane order at the level of the immune synapse, and this is sustained over a period of time, and this is what is quantified here. Not only that, but also at the level of reorganization and redistribution of proximal signaling molecules like the tyrosine kinase LCK, we also observed changes, and you can see here the cells exposed with LXR ligand show a more peripheral distribution of LCK compared with a focal distribution of this molecule in the vehicle-treated cells, and this is what is quantified here in this panel. In addition to the distribution of these cells, we also saw changes in the expression of overall phosphotyrosine levels and the expression of these LCK molecules. So there are clear changes in the stimulation and enhanced proximal signaling in these cells. And finally, we saw these changes were associated also with changes in the production of certain cytokines, and here I'm showing you IL-4 and IL-2 that are important for T cell function and T cell differentiation, whereas other cytokine service, such as IL-17, are reduced. And this is, again, associated in activated cells with a reduction in the proliferation of these CT-4 positive cells. So I hope I have convinced you that in addition to the more established roles in whole body lipid homeostasis, these receptors have also a role in lipid metabolism, not only in macrophages, but also in T cells, and not only at the level of cholesterol and phospholipids, but also impacting the glycosphingolipid changes, and this has an impact at different levels in immune cell function. And we're now going beyond that, and we're looking at things in macrophages, where we saw similar changes as we have seen in T cells, although it seems that the glycosphingolipid changes that we see here in these monocytes are not due to changes in the expression and activity of UECG, because we don't see a regulation of this enzyme by the LXR ligands, and this is the work of a PhD student, Annalisa Maio, in the lab. So over the past two decades, almost, the group of Liz Jury has been looking into how altered membrane cell lipids are associated with immune cell dysfunction, and this is mainly in the context of autoimmune diseases, such as lupus. So in addition to lupus, we also turn into multiple sclerosis, which is another autoimmune disease, and the reason why is because we saw that LXRs were affecting pathways that were important for the function of different cell types that are key in the pathogenesis of this disease. So we checked the target gene expression of the genes that we had identified in our CD4 positive cells, and we saw that in a form of multiple sclerosis, that it's a relapsing remuting MS, we saw clear changes in the regulation of these LXR target genes, and for example here I'm showing you one of them, again, the UGCG enzyme, and this is associated with changes in glycosphingolipid levels in the membranes of these cells. And we think that there's an interplay between the lipids in the circulation and the lipids of the membrane, and these are going to affect changes in immune cell function and eventually promote disease. So we've been fairly successful at looking at metabolomic profiles in different autoimmune diseases, including adult and juvenile forms of SLE, but also multiple sclerosis. Another PhD student in the lab is actually looking at the profiles that we've seen of the lipids in the circulation and how they're related to changes in the membrane lipids in these immune cells and how they can affect the progression of a disease such as multiple sclerosis. And I have to thank now, I think I've mentioned most of the people that were involved in these studies. We have many collaborations over the years that, as I say, they're the true immunologists. I have also to thank the various funders that were critical to fund these studies. Of course, all the patients and the healthy donors that very gratefully donated their time and their bloods for our studies. We published a book, or we edited a book a few years back where some of the, one of the chapters is dedicated to explaining more in detail some of the methodologies that I've explained today. And just to finish saying that, you know, I recently moved from London, after almost 15 years there, to Spain, in the south of Spain, in Seville, where I'm starting a new lab, also looking into LXRs and cardiovascular risk. So if anyone, and if there are any trainees that are interested in doing nuclear receptor work in the south of Spain, in this beautiful city, please come and talk to me. So thank you very much for your attention, and I will be happy to take any questions. Thank you. Great work. So we're open for questions. I'll start off, and I'm interested in the synapse data you showed and kind of the kinetics of that. So if you pre-treated your T cells and then looked at their synapse, does that impact how they're activated, or does that impact what's recruited to the synapse? So we have to, I mean, we need to activate the T cells and engage the TCR anyway, because otherwise we wouldn't be able to see immune synapse. Right. So that's a prerequisite. And then we treat the cells with the LXR ligands. So we don't, we haven't done immune synapse studies without engaging the TCR. Yeah, no, no, no. I just, because you showed data of both sort of naive T cells and then activated T cells. Yeah. So I was wondering if you pre-treat and get that already disrupted cell membrane, and then activate them, throw them on the synapse. Yeah. So I think most of these studies actually pre-treat the cells with, we are pre-treating the cells with LXR ligands. Got it. And then, so you, I'm sorry, I'm going to take everyone's time. The, so you showed a couple things about the synapse. Did you look at other things like PD1, CTLA4, any of the checkpoints? No, no, but we have, we're seeing changes in this, in the expression, at least mRNA expression of these molecules with LXR ligands, yeah. Thank you. Last one, very nice work. And it's a question about the ligand treated state versus the in vivo activation. So if you compare the range of activation, which you achieve with the ligand to the various states in, from patient samples, how does that compare? In terms of? Do you ever reach that level of activation in vivo, then what you can produce in vitro? You mean in terms of target gene expression? Yeah, for example, yeah. So the thing with the level of expression in diseased patients, whether it's multiple sclerosis or SLE, we can, yeah, we can compare to, you mean compare to healthy controls, so no, yeah, the changes are smaller in in patients compared to the pharmacological activation that we can see in In in the in the cells But I think it depends as well on which targets and I would probably go as far as to say it depends on which cell Subset we're looking at so as you know LXRs are extremely I think context dependent so Some of the things that we're seeing in monocytes We don't see in the T cells and some of the things that we're seeing in the T cells in terms of the activation We don't see in the monocytes, but I think to answer your question Overall the probably the changes in terms of target gene expression and are lower In the patients compared to the pharmacological activation of the receptor Thanks Ines, I am from Illinois. So I was fascinated by this new target UGCG you identified and showed So in your patients can is there like a sex difference in response to UGCG or if their diet is different because LXR Can be modulated. Do you see any of those? Yeah, that's an excellent question. So actually we haven't done So most autoimmune patients are going to be women It depends on on which communities we're talking about though, so in the terms of LSC sorry, SLE the The ratio is 9 to 1 women to men So the vast majority of the samples that we have are women in multiple sclerosis is not that much But there's also there are also more women than men. We haven't studied whether there's a Difference in the regulation by by sex no or by sex hormones And the diet the other diet question is also good. It's we Give questionnaires and we're trying to dissect What what whether the diet these patients are on are different and we actually have a program not in MS but in SLE looking at how changes in nutrition could affect the Severity of their disease I actually have a few questions So I mean in terms of looking at the sex differences if you would go and take cells from like IBD patients It's more of a one-to-one ratio. So that would pretty quickly Tell you whether there's gonna be sex differences just saying but okay The other question I have is that so you're pre treating yourselves and then you're looking at TCR activation Do you think that you could possibly dissociate that if you would? With further downstream activation of the T cells if you would treat the cells like maybe an hour activation Like do you still think you'd still see the same downstream? responses of changes in cytokine and Yeah, that's a good question we pretty much stuck to Treating the cells and Engaging the TCR with c3 and c28. So that's that we I think we have done those experiments Okay, so I would basically tell the difference between like what's being activated by LXR the TCR per se and then like downstream responses Which I think would be interesting to tease out as well Yeah see you showed that Cholesterol staining in the membrane was was being altered and I was just wondering so there a group at U of I Chicago is looking at inner leaflet versus outer leaflet cholesterol And I was wondering if you'd considered that particularly since you're engaging a synapse with the outer leaflet. Yeah. No, that's a good question as well No, we haven't done that And one last question have you looked at alternate ways to activate T cells My curiosity is almost Satisfied So you you activate with cd3 cd28. Have you tried activating sort of downstream of that with PMA and I know I know my son So some of the immune Synapse formation studies, I think the one of the of the membrane order with the Super resolution microscopy was done also activating the cells with the PMA. Yeah, okay. Fantastic All right, thank you very much All right This is just a public service announcement Remember today at the end of all of our sessions around 545 the endocrine Basic science reception will be out here somewhere so Everyone's invited if you're not a basic scientist, you're in this room So now we've adopted you as a basic scientist, please join us for a reception and our next speaker is David Moore from now Berkeley and he's going to be talking about nuclear targeting in inflammatory bowel disease Is All right well I'm also gonna add my thanks to the organizers for the opportunity to come back in person to Endocrine Society where I've been coming for many years and As some of you know, I've only recently moved to UC Berkeley and started my lab there about a year and a half ago And as a few of you know I often start my talks with a photo of the location of the where the meeting is instead of what people usually do which Is like this, right? So usually people have a picture of their home institution or something like that So I couldn't find a good picture of Atlanta, but instead I have a picture that I took for my backyard This is for real Okay, so I don't know if it's really Complete search or not, but when I when I searched yesterday Via the app for inflammatory bowel disease. I couldn't find any other presentations or posters Maybe maybe I missed something but there's nobody else talking about an inflammatory bowel disease I could find You know via that anyway, and so I should give a little bit of introduction. So it's an autoimmune disease of unknown etiology and it's relatively common a hundred or two hundred per hundred thousand and It's a it's a pretty serious disease that has a high morbidity and it's it's associated with you know, pretty unpleasant symptoms of weight loss diarrhea fever gastric dysmobility and It's divided into two major types There's Crohn's disease that can occur anywhere in the intestinal tract and ulcerative colitis which as its name implies is more focused on the colon, right and It's treated typically by a variety of the anti-inflammatory strategies that we have therapeutically But none of them is really curative and you know So standard treatments like glucocorticoids and things like that do tamp it down But but they they don't they don't get rid of it and it's it's a chronic disease. It's actually quite unpleasant and I should say that the reason that guy's name is up there is Because he was first described by the Greek physician Soranus who had does have an unfortunate name in this context All right, so a little bit more about inflammatory bowel disease so you can see then on the left that Crohn's disease is going to affect different places in the the whole GI tract and you focus a lot today on the duos or the ileum where There's a particular focus of this disease whereas Ulcerative colitis as you can see is actually more more focused towards the the terminal end of the of the the large intestine There are multiple mouse models of Inflammatory bowel disease. None of them are really that great There are two primary Pharmacologic or chemical models TNBS is a really sort of nasty thing where you actually hapten eyes The large intestine basically by squirting in TNBS from behind DSS is something that is added to the drinking water and it decreases the Mucosal barrier and and so it then compromises that and allows inflammation to come via that way the more complicated but somewhat better model uses The so-called T cell transfer model and in this case what you do is you have immune compromised mice severely immune compromised mice and you treat them with T cells that have basically been purified away from T regs and are then unrestrained once they're injected into these mice and presumably there are other targets as well, but they Focus on the on the intestine and they they induce an inflammatory response throughout the gut or mainly in the colon actually And This is the way that works all right, so you take the purified T cells, right and you inject them into a rag to recipient and You wait a few weeks So it takes a while for this immune response to happen and the way that these experiments are typically read out is relatively simple just in body weight of the animal and Depending on the circumstances the wild-type mice or the control mice I should say are going to be gaining weight whereas the You know the T cell treated mice will either stop gaining weight or in some in some circumstances have pretty severe weight loss and this is associated with Some inflammation in the ileum and also in the proximal colon as you can see measured here with TNF alpha and IL IL-1 beta and IL-6 So this story from my lab starts with a phone call that I got from Mark Sundrud who is a real immunologist at the Scripps, Florida and he had been interested in the question of what happens to T cells in Different places and this is a sort of a burgeoning. I'm sorry So he had been interested in T cells in different places, right and He came across a story that I'm going to tell you today that relates to Somewhat unexpected actually for me quite unexpected function of CAR in T cells in the intestine So what is CAR? So CAR is a xenobiotic receptor That's well studied in the liver and along with its close relative PXR so CAR and PXR are sort of like ER alpha and beta that are each other's closest relative, right? and they both function in response to xenobiotics, which are foreign compounds that we're exposed to and The liver has the well-described capacity to induce drug metabolism in order to clear such foreign compounds away, right? so one of the functions one of the many functions of the liver is to do that and CAR and PXR are the two receptors that do that. They do it slightly differently. CAR is primarily responsive to so-called phenobarbital like Activators that induce various sorts of stress in the liver. Of course, it does have direct agonists as well and PXR is actually more responsive to a Wide variety of different drug inducing agonist ligands that are foreign compounds Now both of these receptors are also responsive to endogenous toxic molecules Including bile acids and PXR is directly activated by bile acids and CAR is apparently indirectly activated by toxicity of bile acids And another thing to remind you those of you who are nuclear receptor aficionados will be well aware of this but Others maybe not so much. So bile acids are ligands for the nuclear receptor FXR They're also of course Functional in digestion, right they dissolve nutrients and and lipids in our diet and allow us to absorb them, right and in that context they're made in the liver, of course, right and then into the gallbladder and then into the small intestine right where they they promote digestion and As part of that process when the bile acids get to the ileum at the C term at C terms at the distal end of the At The distal end of the intestine small intestine, right? They're actively reabsorbed in 95% of them go back to the liver via the portal circulation, right? So this is a tightly regulated process FXR is negatively regulating bile acid production in a classic sort of endocrine feedback loop, right? so It's critical then that the the ileum is the site of Reabsorption of bile acids All right, so in the context of car activation and PXR activation by bile acids We found years ago now that car seems to be actually a little bit more sensitive to at least the car knockouts I should say a little bit more sensitive to toxic bile acid. So lithicolic acid is a nasty one That's more hydrophobic than normal bile acids And if you treat mice with that it induces liver damage in a variety of which different ways and the car knockouts are much more Sensitive to that than the PXR knockout. So this car really does regulate it protect us against bile acid toxicity And as I've already mentioned it turns out that for reasons that were not really very clear a substantial fraction of patients that have Crohn's disease It affects the ileum. So this is actually from a patient who has ileal colitis and it's you know, narrowly directed towards the The ileum and about 25% of Crohn's patients actually have ileal targeting So as I said Mark was interested in the idea of you know, what are T cells doing in different contexts So, you know, we were just hearing about T cells in circulation and things like that which of course is where we ordinarily study them, but T cells are present in a variety of different tissues and have a variety of different functions and They, you know, they move into different tissues they can be activated or not and have T effector functions in different tissues and they have a variety of Functions in different tissues. And so Mark was interested specifically in T cells in the ileum and What he showed in this paper From a few years ago is that the T cells in the ileum have gained something unexpected Which is expression of the drug efflux pump MDR-1 Right. So MDR-1 as you may know in the context of cancer and drug treatment Is a drug efflux pump that removes toxic things from cells, right? And in the context of the T cells in the ileum what Mark showed was that MDR-1 is actually necessary For them to protect themselves against the high levels of bile acids that they encounter Through the flux of bile acids that's reabsorbed in the ileum So the bile acids are meant so these little red dots are the bile acids, right? So here's the T cells So what you can see here is that in MDR-1 knockout mice if you do the T cell transfer experiment What you can see is that there's a more severe weight loss, right? So the the MDR-1 mutant T cells, defective T cells are experiencing for additional inflammation because of the stress of the bile acids, right and That's shown here also with the numbers there's The number of T cells basically when you when you look in the the mutant transferred mice There's actually quite a few quite a few smaller number of T cells And this difference is prevented by the bile acid sequencing sequestrant cholestyramine so cholestyramine is something that is used in dietary dietary mode to actually bind actively bind bile acids and Doesn't let them get into the you know into the the intestine back into the intestine All right, so Mark was clever guy and what he did was a screen an RNAi screen where he treated the T cells before they're reintroduced with a pooled library of Targeted library that had all of the nuclear receptors and a few other things a few co-regulators The aryl hydrocarbon receptor was in here and things like that negative controls and things like that So what he found when he read that out is That there were several receptors actually that Ended up on this end of this graph which means these are the ones where the The T cells had quite a lot less expression of MDR-1 and it wasn't actually the top one But one of the ones that was there is CAR, right? so He actually contacted me About that because we work on CAR, right? And so he you know was asking me about getting some mice and things like that And when he explained to me exactly what had gone on, you know in the you know to lead him to do this experiment I said, well, wow, that's fantastic That makes perfect sense because you know, we had already been studying the you know The toxicity of bile acids in the liver. So why wouldn't it happen the same way in T cells, right? And so we started working with him So what we found with Mark is that if you purify the T effector cells from the small intestine and You just look to see whether CAR agonists cause induction of CAR targets. The answer is yes, so CAR is expressed there MDR-1 is expressed there. CYP2B10 is the the top induced target in the hepatocyte and it's also induced in the T cells and importantly IL-10 is also Induced in response to CAR activation in these T cells and as many of you will be aware IL-10 is a anti-inflammatory Protein that is actually quite important in the context of inflammatory bowel disease So if you look in vivo for what happens when you do the transfer not with the MDR knockouts But with the CAR knockouts, it's basically exactly the same result, right? So I actually originally thought that this experiment that the CAR knockouts might be Protected because the T cells would just die and there wouldn't be any immune response But no instead they become basically as I said, you know, they're more pissed off, right? They're more unhappy, right? and and so you can see that you know just like before there's there's a decrease in body weight and there's also And you can maybe see that the villi in the ilium are Shorter and there's a you know disease and activity index that is not apparent in the no effect apparent in the colon But in the ilium you can see that there's a more severe disease with these mutant cells And An important part of this that we don't have a lot of detail on beyond this is that the IL-10 messenger RNA is is definitely up and If you do this with RNA-seq from the isolated T cells, you can you can see that there's quite a big drop in the small intestine T effectors and also the colon colon precursors and This is a IL-10 reporter That is also decreased in these cells. So there's a functional drop in IL-1. Sorry IL-10 So what about the opposite right so what about car activation? sure enough if you You know treat the cells with if you treat T-cell transferred mice with a car agonist the TC compound that we use which you can see here Let's go through this one first So these are the normal mice and they're you know, not not changing their body weight very much if you So The the chow vehicle So these are the mice that are on the chow if you give colic acid to their these chow T cell chow chow diet Sorry, if you give colic acids to the T cell transferred mice the this they do much worse But if you take those T cell transferred mice with colic acid and give them the TC compound that activates car It brings it quite a bit back up, right? What's shown here is it's a little bit complicated but so that we're showing the response to TC so there's a like a fourfold response higher response of CYP10 CYP2V10 Right, so these things are responding to the TC compound here in the wild type transferred cells But in the mutant transferred cells the response is gone, right? This is as expected and again there's actually an improvement in the, sorry, the TC treated cells have an improved disease response as expected. The last result here is that there is a characteristic signature of the T cells that are present in the small intestine, the homing, there's a homing mark for them. That's this, yeah, alpha four, beta seven, CCR nine, right, so a double mark that is indicative of cells, characteristic of cells that are in the small intestine. And if you sort human cells from the spleen for the presence of that marker, those are cells that are presumably on their way out of the ileum, or the intestine anyway, and are gonna, we think, retain some of the marks that they had. As you can see, they do retain expression of MDR1, and CAR, and the human analog of 2B10, and some elevation of IL10. So it looks like this is a phenomenon that's probably also present in human T cells. So this is the conclusion of this part, that I didn't go into it, but we believe that there are other biliary metabolites that are not bile acids that are somehow involved in this process, and we don't know what those are. We have evidence for them, but we don't know exactly what they are. But it's clear that CAR is doing in these T cells more or less what it does in hepatocytes, which is to protect them against bile acids, which, as I told Mark when he first called me, makes just, makes a lot of sense. It's a question of how they express, so the first question is, why do they express MDR1? Oh, they have CAR. So why do they express CAR? We actually think that that's due to an FXR function in dendritic cells, but we're working on that. So another brief part of the story is what we were actually working on in the context of inflammatory bowel disease before Mark called. And this is a story that Christina Schoonjons and colleagues in Switzerland had developed over years, and what they found was that LRH1, which is the alpha beta thing, just like CAR and PXR are alpha beta, LRH1 is the alpha beta of SF1, right? And in the adrenal, SF1 induces formation of adrenal steroids, of course, from cholesterol. In the liver, LRH1 induces the expression of bile acids from cholesterol, so they have sort of similar functions. And in that context, it's surprising that in the intestine, there's a pathway where the steroidogenic enzymes that are induced by SF1 in the adrenal are instead induced by LRH1, and that happens only in the context of inflammation. So in the inflamed intestine, what happens is that LRH1 drives a sort of negative feedback anti-inflammatory pathway of local glucocorticoid production, right? In CYP11A1 and CYP11B1, the steroidogenic targets are LRH1 targets in the intestine, as they showed very elegantly. And they also showed the LRH1 straight knockout is lethal, and so the heterozygote, for example, has more sensitivity to the mouse models of inflammatory disease that I've already mentioned. Right, so that raises the question of whether LRH1 activation is good, and I'm not gonna go into this in a lot of detail today. We're just gonna show you a couple of slides, but the, so we have done all three of these models with LRH1 activation, and the answer appears to be yes. There have been some issues with getting this to work reproducibly over a relatively long time, but we're pretty confident that we've recently sort of restarted and gotten it to work, and so we're confident that it does work, but it's difficult to work with the agonist ligand that we have for LRH1, which is called DLPC. DLPC is dilaroyl phosphatidylcholine. So this is sort of like a membrane phospholipid, except the fatty acid side chains are short, C12, right? And so this is actually used to make liposomes in therapeutic context, actually, but so DLPC does act as an agonist ligand for LRH1. There's a crystal structure of LRH1 bound with the DLPC that was solved by Eric Orland, but what you can see here is that if you treat the mice with DLPC by intestinal, by, sorry, by intraperitoneal injection, that it has a pretty beneficial impact on the inflammatory bowel disease process, right? And you see, of course, that there's induction of CYP11A1, which is the sterogenic target. TNF-alpha goes down on other inflammatory markers. There's a twist, is actually a transcription factor that's more associated with other pathways, but in this context, we think that what's going on here is that twist is known to activate the expression of glucocorticoid receptor itself, and so there is at least a modest induction of GR. And so what's diagrammed on this slide is actually the combination of all those responses, which is a sort of a feed-forward loop where LRH1 activation not only causes production of glucocorticoids, but also via twist, and we have some genetic evidence for this, induces expression of GR itself. So the combination of this is a coherent feed-forward loop, and this results in a more stable induction of an anti-inflammatory response. So the story that I wanted to tell you, and again, we've done this experiment once, but I'm so excited about it, I decided that I would tell you about it anyway. It's a little bit complicated. So Kevin Klatt is a PhD RD, and he got his PhD RD at Cornell, and he had an idea that he then, so he, this is another phone call, so he called me up, and he told me, you know, I think that if you treat cells with C12, which is lauric acid, right, laurate, right, if you treat cells with laurate, the phospholipases and acyltransferases in normal cells will actually allow them to make their own DLPC, and you don't have to go through the problems with delivery of DLPC, you can just do laurate. And I said, Kevin, I don't think that's gonna work. But it turned out that he had already done it. And so half of his thesis was that. He actually even did that, so he, you know, he comes from a nutrition background, so he actually gave people milkshakes that had laurate in them, and measured DLPC in their blood. So this happens actually in people, right? So the question is, if you treat these, you know, mice with laurate, does that work? And the reason that the coconut is up there is that, so laurate is actually not a very commonly encountered fatty acid, right? So as you know, fatty acids are degraded two by two and built two by two, but they don't stop in the middle, right? And so the question is, so are there natural products that have abundant laurate? The answer is yes, coconut oil is about 50% laurate. So coconut oil is actually an abundant source of lauric acid. It's actually contentious whether laurate is good or bad for you, because it's a saturated fatty acid. So some people think that it's God's gift, and other people think that it's the devil's tool. So Kevin is now with me at Cal. And we did this experiment in Houston, but as you can see, if you measure the body weight of mice that are treated with either corn oil, right, or coconut oil, there is an improvement in body weight. So it looks like, you know, that the coconut oil may actually have a therapeutic benefit in the context of inflammatory bowel disease. And you can do various ways to, you know, look at the impact of disease. We, of course, look at target genes too, but there's, you know, you can get scores or weight loss, diarrhea, hematochezia. This is blood in the stool, and, you know, disappearance. And so there's a disease activity index that's the sum of all these things, and you can see that it looks like there's a beneficial effect. Like I said, take this with a grain of coconut oil. We, you know, we need to confirm and expand on this, but I'm excited about this. So that's where we are. We think that targeting CAR, particularly in the context of ileal Crohn's disease, is an effective therapeutic strategy, and there are a number of human, you know, compounds that will activate human CAR that are potentially useful for this. Artemisinin is one, right? It's metabolites anyway. And also we think that targeting LRH1 to drive local glucocorticoid production is also a viable therapeutic strategy for inflammatory bowel disease. And with that, these are the members of my lab. On the left are the ones that were in Houston. Jay Monn is the one that started this whole thing, the inflammatory bowel disease story, with LRH1, actually. And Shang Sheng is the one who did most of the work in our lab with Kevin and with Guo Wei on the CAR story. And that wouldn't have happened at all without Mark, and Mei Lan is the graduate student in this lab who just graduated not that long ago, who actually was the real main driver. So Shang Sheng moved to Cal for a while, and he's actually back in Houston, but Kevin and Lindsay are gonna be moving forward with this. And my lab, as it's struggling to get off the ground in Berkeley, will, I hope, be able to do some more experiments to expand on these interesting questions. And with that, the light just turned red, so I think I'm done. I saw that you have YAR alpha in the, I thought you would notice it there. And last year we published a paper on with the DSS model using the knockouts. And one of the problem there was, the main problem was a change in the diversity of microbiomes. And I wonder if in your models and with different treatment that you give. Right, so the microbiome is obviously an important player here and so far, you know, an important issue that we haven't addressed. Okay. David Shapiro, Illinois. So for about 20 years, people have tried treating cancer patients with inhibitors of MDR-1 and they've all failed therapy mostly because of severe side effects. Is one of those effects the activation of this pathology in the intestine? You know, I'm not familiar with that and that's an interesting possibility. I mean, the, you know, there are degrees of this sort of, you know, intestinal, you know, problem. So frank inflammatory bowel disease is one thing, but a lot of other people have, you know, intestinal symptoms that are unpleasant, you know, that come and go. So I wouldn't be so surprised. The, I should say that the CAR knockouts don't have an obvious, you know, basal ileal inflammation. Sam Jerzak from Emory, actually Ortlund Lab. So nice to see you. I noticed that in the screen that Mark Sunder did for MDR-1 regulation, actually one of the things that was best at down regulating MDR-1 was also LRH-1. So have you explored the roles of LRH-1 in modulating the immune system and outside of just an indirect effect on glucocortisol? So we haven't done that, but what's her name? There's a lab in Europe that has done some work on LRH-1 in macrophages and there are some functional impacts, yes. Actually, I can answer, sorry. Mark's my colleague, so his lab is right next to mine. But actually, so LRH-1 knockouts, people have looked at different immune cells in it and there's definitely effects on CD4s and CD8s and pretty much most of the immune cells that are, have been looked at. So it's not just, it's not just macrophages. Hey David, Carlos Bernal from Washington University. What will be the expression of CARs in other immune cells and interactions with T-cells here? Is the CAR also critical in macrophages and the interaction of T-cells in IBD? We haven't really found much CAR expression outside of the ileal T-cells. And I wouldn't say that we've looked everywhere. So we don't have that. The only other place that I'm aware of is that I've been told that immune cells in the inflamed liver do have also some CAR expression. But that's a, you know, more narrow circumstance, right? So I'm not aware of functional impacts of CAR outside of this circumstance. So I'm just, oh, sorry. I'm thinking of how to drug that. Do you think it's possible to hook up a CAR ligand to something to make it so it can't be absorbed and you just take it orally? Would the intestinal T-cells get enough exposure from that? If it's not absorbed, it might not really get in, I don't think. But the, I mean, I wouldn't worry too much about absorption. So CAR activation is not necessarily a terrible thing. Right? Of course, you would have to be very careful about other drugs that you were taking if you were in that sort of circumstance, right? The, but, so phenobarbital, which activates CAR, was used for decades for, you know, seizures, right? And it actually does have metabolic impact on the liver that is related to its, you know, sort of a side effect of its CAR activity, CAR activation. But in terms of other sort of negative outcomes, not so much. So I think that CAR activation is probably okay. Okay. I have a quick question. So in your model that you showed where I think you said you want to repeat it, the corn oil versus the coconut oil, are you planning on just putting, like, normal T-cells in that with no treatment to see whether the corn oil is, like, exacerbating disease at any, through the T-cells? So I think that would be interesting because, I mean, just given the way that the corn oil and coconut oil are metabolized differently, it could, you know, have profound effects on not just the microbiome but on T-cell metabolism as well. Right. So as a nutritionist, Kevin is actually very, you know, attuned to all of these kinds of things. And, yeah, for sure, all of us, yeah. So you need to, you know, you have to have controls for the controls in these nutrition studies. And he's actually shocked by the sort of high-fat diet experiments that most biologists do for, you know, metabolic disease and how they're not really controlled, you know, correctly in all kinds of things. Okay. Are there any more questions? If not, thank you again, David, for a great talk. Thank you. If you want more on IBD and T-cell responses, my colleague tomorrow is giving a talk early morning, is it? Yes, I think it is. So wake up early and get more. Right on the bushy tail. So our next speaker is Shannon Dunn from St. Michael's Hospital. And she's going to be telling us about PPARs and T-cells. Hi, everybody. I'm really glad to be here and glad to be here, really, because I had a hard time getting out of Toronto last night. I rolled in quite early this morning. And I don't know, my brain was not firing on all circuits this morning. And I somehow convinced myself this session started at 9.45. But I'm here, so that's really great. So I just need to know how to afford slides and stuff. Okay. I can figure this out. I've had two coffees now. It's starting to kick in. So I really want to thank you again for the invitation. So where do I come from? Well, I'm actually a neuroimmunologist. I'm not a nuclear receptor person, but I'm a neuroimmunologist who developed an interest in nuclear receptors. So the disease I'm interested and passionate about is multiple sclerosis. It's an autoimmune disease, a T-cell-mediated autoimmune disease that targets central nervous system myelin. So these patients develop lesions in the brain. If we kind of drill in and kind of put a microscope on this brain, we're going to see that there's, not sure where the pointer is here, but it's okay. If the top right, we're going to see macrophages, very angry. And in those areas, we have loss of myelin. And the macrophages are angry. And you can see they're particularly clustered around blood vessels because that's where the T-cells come in. So this is a blood vessel, our venule in the brain. And through many experiments, it's thought, although we don't completely understand the ideology of MS, we have an appreciation that both T-helper 1 and T-helper 17 cells of the pro-inflammatory phenotype are important in probably getting these attacks started. So I got an interest in nuclear receptors when I was doing my postdoctoral fellowship at Stanford. I was in Larry Steinman's lab, but just down the hall was a guy named Dr. Ajay Chawla, who was studying the PPARs in the immune system. And he had just had a beautiful paper in cell metabolism where he described a role of PPAR delta in promoting M2 polarization of macrophages. So I was really excited about these molecules and I got some tools and reagents from him and we started collaborating in the animal model of MS. So I guess I don't have to spend too much time telling this audience about what PPARs are, but just as an overview, they're members of the nuclear hormone receptor superfamily. Just like all nuclear transcription factors, they both activate and repress gene expression, which makes them all so complicated. And there's three family members, alpha, delta, which I'm talking about today, and gamma. And what you may not appreciate is that they all have very important roles in controlling T-helper cell function. So previously, our group and others have described a role for PPAR alpha inhibiting T-helper one biology, particularly in males, there's a sex difference in that phenotype. PPAR gamma seems to control TH17 responses. And PPAR delta, which is the most ubiquitously expressed molecule, has a bit more of a complicated role in T cell biology, which I'm gonna describe to you today. So the first experiment we did with Ajay is we got a hold of his PPAR delta mutant mice. These are the mice generated by Jeff Peters. So they have a neocassette targeted to the ligand binding domain of the receptor, which results in essentially a very kind of an unstable transcript and null protein expression. So what we basically did is we induced the animal model of MS in these mice, and it's essentially a vaccination model. So just everything I'm talking about today, it actually has relevance to just vaccination in general. So we immunize the mice with a piece of myelin antigen, in this case, myelin and leukodendrocyte glycoprotein, and complete Freund's adjuvant. And after about 10 days, these mice develop an ascending paralysis, which we have a scale for, you can see in the graph on the left. And what we found was that the wild type mice had more of a relapsing remitting form of the disease, which is kind of what we see in MS patients. But those deficient in PPAR delta just developed more severe EAE. And when we took a look at the spinal cord, which is the top right picture, we see a lot more inflammation in the CNS white matter. So at the time, we also characterized in greater detail the profile of immune cells in the central nervous system using flow cytometry. And we had just seen that there was just a lot more pro-inflammatory Th1 cells in particular, but also a tendency for more Th17 cells in the CNS. So this was right when I started my lab. So this is my first crop of graduate students. And I put a few of them to work to try to better understand what PPAR delta could be doing in the immune system to protect against EAE. So Paulina there, she kind of tackled some of our myeloid cell phenotypes. And Linda on the right is, that's the work I'm gonna present today. She tackled the role of PPAR delta in T cells. So we just started doing very simple experiments where we isolated T helper cells using magnetic beads or fax sorting, and we activated them in a dish with anti-CD3 and anti-CD28. And it sounds like our first speaker was introducing this assay, so I won't describe it in big detail, but this is just gives the T cells the signals to proliferate and produce cytokines. And as a new professor with your first grad student, I was a little bit scratching my head because this is the type of data my graduate student was bringing to me. So half the time she would do these experiments and on the graph on the left, she would find that P-par delta knockout cells actually produced a lot more cytokines compared to wild type. But the other half of the time, she came to me with data to suggest that they actually produce less cytokines. So what was going on here? I had to actually follow her through the experiments to make sure she was labeling the tubes, right, and all these things. But it ended up being that it was, after looking at her lab notes, it was depending on what reagent she was using to isolate the T-cells. So she's using a kit that would just isolate all the T-helper cells, or if she tried to isolate naive cells but the purity wasn't great that day, she would get a phenotype where the knockout T-cells were producing more cytokines. But it kind of, the phenotype flipped when she was actually able to get good purity or fact sort naive T-cells. So again, starting with a very complicated phenotype, but we actually were able to kind of figure out what was going on. So the first thing is that Linda discovered is that PPAR-Delta has a very important role in supporting thymocyte development, so a T-cell development, and it actually, in these knockout mice, these T-cells don't do as well in thymocyte development, and because of that, they actually have a lymphopenic phenotype. So in the periphery, when you don't have T-cells coming out of the thymus as well, the ones that are there turn over and become memory cells which are hyperactive. So when she was using those beads where those contaminating memory cells were present, she would get a lot more inflammation. However, because of this metabolic phenotype, PPAR-Delta knockout naive cells, actually, they needed kind of a more robust metabolism to support their proliferation, so when she was just isolating naive T-cells, they didn't proliferate as well or produce cytokines. So it kind of explained our phenotype, but we also discovered that PPAR-Delta itself also controls both Th17 and Th1 inflammation in the T-cells. So there's really three different stories, and I'm just gonna try to kind of give you the broad brushstrokes today. So let's just start in the thymus. So this cartoon shows kind of very briefly how thymocytes development. So they start in the bone marrow, and the precursors travel to the thymus, and there they undergo a variety of different steps to develop into what we recognize as CD4 and CD8 T-cells. And it's at this DN3 stage of thymocyte development where they first up-regulate or rearrange their TCR beta chain, and that TCR beta chain pairs with kind of a generic TCR alpha chain, and it's at that stage the T-cells get their first signal, and they can start to undergo this large proliferative burst. And in fact, they actually divide about eight times at this stage of thymocyte development, which is really important for creating TCR diversity at the next stage. And it had been recognized in the past that there was a really important role for aerobic glycolysis, up-regulation of glucose transporters, transfer in an amino acid transporters to kind of support this process at this stage of development. So when Linda tried to figure out where the block in thymocyte development was in these knockout mice, she, again, interestingly saw that the drop-off in cell number started to occur at the DN3 stage and was significant by this DN4 stage of development, so it corresponded to this TCR beta selection step. And interestingly, just in wild-type thymuses, she noticed there was an up-regulated expression of PPAR delta at this stage of development. When we kind of looked at all the kind of the usual suspects that had previously implicated to be important at this stage of thymocyte development, we found everything was normal, including in vivo DNA synthesis, suggesting they were making DNA just fine. So we were really stumped. We just didn't know where the thymocytes were going at this stage of development. It was really tough to address in vivo because when thymocytes die, they kind of get gobbled up by macrophages very quickly, and you can't really examine cell division in vivo in a mouse. So luckily, in the Department of Immunology, where I'm a faculty member, there was a guy who has a in vitro thymocyte differentiation system called the OP9 Delta-Leg4 system. This is Juan Carlos Zuniga-Fluker. And essentially, he has these nice thymic stromal cells that he has in a dish, and you could seed these DN3 thymocytes onto these thymic stromal cells, and then watch, and they can undergo all the different stages of thymocyte development. So when we sorted wild type and these PPAR Delta knockout thymocytes and put them in this system, and these were labeled with a dye called CFSC that we can therefore track cell divisions, we are able to see on the bottom left graph, you can see these DN3 thymocytes do differentiate into all the different thymocytes we need. And again, we start to see a drop off at the DN3, DN4 stage. And it was because these cells were unable to divide as well. So they seemed to, they weren't dying off in greater amount numbers, they were actually unable to divide. And they also, we also had a tendency for them to be smaller. So suggesting there was a defect in these cells to generate enough biomass to undergo these repeated cell divisions that were required at this stage of thymocyte development. So we did have some transcriptomic data from the PPAR Delta mutant T cells. And there were a lot of metabolic genes that were downregulated in these cells. And we validated that these same genes were also showed defective expression in our DN3 and DN4 thymocytes. So essentially, we found that without PPAR Delta, you had decrease in expression of genes involved in, I'm not sure this is the pointer, here's the pointer, I now know how to use it. So second cup of coffee is kicking in. So we have defective decreased genes in glycolysis, ability to, genes involved in transporting pyruvate into the mitochondria. We had a citric acid cycle enzyme members that are important in the electron transfer chain, as well as in keto metabolism. So enzymes that can essentially generate acetyl-CoA that can potentially be reshuttled into making membranes as cells divide. And all these genes were downregulated and many of them do have bonafide PPAR response elements in their promoters. Interestingly, the one gene that was highly off the chart upregulated was a chain of hemoglobin. And we had read that hemoglobin is also an antioxidant. So we speculate that this gene is being upregulated to somehow compensate for the defect in mitochondrial ROS production, which is kind of off the chart without PPAR Delta. So just to show that these metabolic defects do impact thymocyte metabolism. So we're able to sort these DN4 thymocytes and put them into what's called a seahorse assay. Essentially in a dish, we can measure the basal oxygen consumption of the cells, as well as their ability to make lactate and in response to feeding them with glucose. And you can see the basal oxygen consumption rate of the knockout thymocytes is lower, as well as their glucose induced lactate production. So the first conclusion of my talk is that it seems that PPAR Delta kind of supports T cell immunity in some ways. Both thymocyte development, as well as I'm gonna show you in a bit naive T cell proliferation, by maintaining a certain basal level of metabolic gene expression in the T cells. So because of this thymocyte defect, it's really hard to interpret any, I guess, experiments that use the whole body PPAR Delta knockout cells because they just develop differently. There could be differences in thymic selection. So it's really kind of hard to interpret this data. And also the knockout mice also have changes in the microbiome and things like that, that further confound interpretation. So we decided to make a mouse that essentially we could knock out PPAR Delta after this step in thymocyte development. And we did that using a Cre-LOX approach. So we use the floxed PPAR Delta mite. This is generated by Ron Evans lab. We crossed it to a mouse that had Cre recombinase expressed under the control of the distal LCK Cre promoter. And this gets turned on late in the double positive stage of thymocyte development. So essentially bypassing this place where PPAR Delta was important. Importantly, these T cells have overtly a normal immune compartment. There's thymocyte is completely, thymus is completely normal, spleen, I'm sorry, I'm sorry. Thymocyte is normal, the spleen subsets are normal. And we were able to detect a knockdown of PPAR Delta gene expression in the peripheral T cells. So Linda wanted just to see if this metabolic phenotype we found in the thymocytes was also true of peripheral T cells. So she did isolate naive T cells and did the same assay where she just stimulates and then dish with anti-CD3 and anti-CD28. And it's very kind of artificial system. But she was able to show that PPAR Delta knockout T cells were not proliferating as well compared to flocks counterparts in this assay. So this metabolic defect does seem to translate to the peripheral T cells. So based on what I just told you, I guess I was predicting potentially when we try to induce EAE in these mice that they'd actually get maybe less severe symptoms. Because if the T cells can't expand as well, maybe they just won't get the autoimmune disease. But in fact, when we induced EAE in the T cell deficient knockout mice in the flocks controls, we actually found that the T cell deficient mice had a more severe form of EAE, which again associated with more inflammation in the spinal cord. So this is a picture of the spinal cord white matter in blue here, we see more inflammation as well as more demyelination. So you can see here the lesions are bigger and in these big holes are actually where axons are being attacked and are dying. When we isolated the central nervous system immune cells out using a PERCOL gradient and characterize them by flow, we did see that just in general, there was more leukocytes infiltrating, more CD4 cells, more T helper cells. But here the story was a bit different from what I told you in the whole body knockout mice. So in the whole body knockout mouse, there's more Th1 cells, which we speculate is probably just there's just more memory cells in that mouse. But in this mouse, we found that there was actually a skew so that most of the cells infiltrating were of the Th17 variety. And there was also a tendency for more pro-inflammatory Th17 cells, which are thought to be the culprits of these lesion formation. We wanted to see if this defect originates in the peripheral immune system. So we kind of repeat the experiment. We vaccinated the mice and induced DAE, but instead of letting them develop symptoms, we take out the spleen and lymph nodes from the mice before onset of symptoms. And then we take these cells and add in increased MOG antigen. We can look at the MOG specific proliferation as well as cytokine production in these organs. And what we found is that the T cells in general, the MOG-reacted T cells are proliferating more. They were making more cytokines. In particular, again, IL-17 was much higher in the PPAR Delta deficient mice. And interestingly, when we just took total T cells, so kind of doing, going back to our original experiment, we saw the same phenotype. So although the naive T cells, the ones that are untouched, haven't seen antigen, are a little bit hypo-responsive, when we look at total T cells, if anything, they're proliferating a bit more, and again, making more IL-17. So what's going on here? It's a bit complicated, but to date we have figured out part of it. And there's actually two things going on here is what we found out. So we think, first of all, we've ruled out that Tregs are working perfectly fine in this mice, but we have found that the naive T cells are actually more prone to making IL-17 so when we push them down the Th17 path, they make more IL-17. And the second phenotype we discovered, if we sort CD44 high cells, they are actually hyper-inflammatory. And so just to remind you that we're starting to drill in in what the real, what T cells are more inflammatory, but this is still a big gamish of different types of cells. So CD4 cells are comprised of Tregs, NKT cells, TR1 cells, naive TFF cells, TR1 cells, naive T effector. So there's a whole bunch of different cells and this is still a work in progress, but I think we're getting closer to understanding what the phenotype. So this is just to show you our Th17 polarization assay. So if we stimulate T cells in a dish with anti-CD3 and anti-CD28, but also add in cytokines that push them down the right lineage, we can see that those T cells without PPAR delta are producing, are becoming more Th17. It doesn't affect Th1 differentiation in the assay. So that's one thing PPAR delta is doing. And this is the experiment with when we sorted CD4. So this is, we essentially remove the naive cells that are hyporesponsive, leaving us with CD44 high cells. We can see when we activate in the dish, they're a lot more pro-inflammatory. They're proliferating more, making IL-2, particularly interferon gamma and some IL-17. So just remember, we're still drilling in, we're still trying to figure out what are the culprit cells, but there's still a number of different cells in this mixture. But it'll be very interesting to figure out. We're gonna now be doing single cell RNA sequencing to try to figure out where PPAR delta is really working. So lastly, I just want to give you with a, leave you with a take home message. So it is complicated. PPAR delta is doing a lot in T-cells, but I think this is how I look at it. I think it actually can be promoting a healthy immune system in terms of kind of maintaining a certain amount of metabolic tone in both the thymocytes and naive T-cells to support their ability to proliferate and start an immune response. At the same time, and in this concept, it seems to be kind of acting as a traditional transcriptional activator. All the genes that we noted in the knockout cells would be down-regulated, were all down-regulated compared to wild type. So we, and they had, most of them had PPAR response elements in their promoters. At the other end, I think it's actually repressing excessive Th1 and Th17 inflammation. And it's doing this in a number of ways. So one way it's kind of promote or inhibiting development of Th17 cells. We think it's actually inhibiting the development of pathogenic Th17 cells. It's acting in some memory T-cell to control their production of cytokines. What I was not able to tell you today is it actually acts in dendritic cells as well to limit the production of IL-12 P40, which thereby also limits Th1 and Th17 inflammation. So, and to remind you that inflammation is known to turn on PPAR delta. So I think it functions as an important negative feedback loop to control kind of excessive inflammation in the host. And last of all, I was just gonna touch on ligands. So all the work I showed you today was in vivo work, comparing a wild type and a situation where PPAR delta was not expressed. In this context, PPAR delta would be occupied by endogenous ligands. And in all our experiments using these synthetic ligands, many of them have very different activities. So I think it's gonna be very important and interesting to identify what are the endogenous ligands in these different tissues and cells to have these activities. Last of all, I just want to acknowledge former members of the Dunn Lab, in particular Linda, who was really driving all this work, as well as new members of the lab. And I want to thank Dan Weiner for his help with seahorse assays, as well as Juan Carlos Zuniga-Fooker for his help with the thymocyte differentiation assays. And thanks for your attention. Thank you. Thank you, Shannon. Actually, I have a few questions. So I noticed that the interferon gamma expression was different in vitro versus in vivo, and you did mention pathogenic Th17 cells. So I'm wondering if PPAR delta is regulating or having an effect on the transition of Th17s into the Th1-like cells, and those are thought to be the pathogenic cells, which would maybe explain some of the interferon gamma results. Yeah, I think from what I understand, I think it's just doing a number of different things in different compartments, but it's true. We're interested in sorting, I guess, Th17 cells, and now looking at their ability to become pathogenic Th17s to see if that is the case, because I think it could be very interesting to further look at that. But I think it's actually working in a CD44, high CD4 cell. I don't know what it is. It could be there's a lot of interest about these new memory phenotype cells or essentially CD44 cells that are inactivated, but can play a role like NK cells in activating immune responses. So I'm interested to see if it could be functioning there to limit that arm of the immune response. In the CD4s that you're pulling out of the different mice, have you looked at if any of those are gamma delta T cells and have you thought about how they might play into it? We just did some, I think in some flow with gamma delta cells, and they did seem to have a slightly more interferon gamma production, but no change in IL-17. So at least we eliminated them as the source of our enhanced TH17 response. And then two other questions. So you ruled out Tregs. Was that just based on number or did you pull out the Tregs and make sure that they were still functional? Yeah, so far we've only done immunosuppressive assays, sorry, in vitro suppressor assays, but they seem to be doing quite well to suppress those. In fact, we, yeah, the ones that were, that's where we kind of discovered the hypo responsive phenotype of the naive cells, but the Tregs were doing just as well to suppress them. They differentiate into Tregs. So if we push them down the Treg lineage, those T cells also differentiate just fine into Tregs. And then my final question is, so you showed in one of the knockout T cells, anyways, there's a disruption of several metabolic cascades. Can you, or have you gone back in and tried to add in sort of downstream metabolites and rescue, if you will? We haven't, yeah, I guess because the, I just showed some of the genes that were down regularly, but I mean, there was so many pathways. I think our next step was to try CHIP-Seq just to make sure these were bonafide targets. So we did get a nice antibody from Jeff Peters, but we never, yeah, got to the experiment. But yeah, I guess there were so many things to target. We didn't know where to focus and how to rescue, but that would have been a nice experiment to be able to do. Hi, I'm Madhur Singh from University of Iowa. I have a couple of questions. One is that TH17 programming, the Rho Gamma T levels, have you looked at the PPR Delta knockouts about the basal level of the Rho Gamma T? And also, PPRs are known to inhibit NF-kappa V inflammatory pathway. Have you looked at that at all in these? I have looked at a lot of things. I'm trying to see if I have, oh, I don't have it. It's okay. I had some, yeah, we did find, there were a number of genes in the TH17 pathway that were up-regulated in the absence of knockout cells. I don't think Rho or Gamma was one of them, but IL-21 was higher. There was probably 20 different genes in that pathway were up-regulated. So it's that the pathway is turned on, and it's hard, but all those experiments were done with the mutant cells. We want to repeat everything now with this kind of cleaner T-cell system. And the other question was, have I looked at NF-kappa V? Yeah, I looked at that. That was probably the first thing I looked at, and there was nothing exciting to write home about, at least in the assay I did, in the cell I did, but I don't remember the details. Thank you very much. Okay, thank you very much. I'd just like to thank all of our speakers for tremendous talks today, a lot to think about, and a reminder about the reception later on this afternoon. Thank you.

nuclear receptors

inflammatory bowel disease

T cells

MDR-1

CAR activation

therapeutic target

PPAR-delta

multiple sclerosis

T-cell biology

immune activation

excessive inflammation

autoimmune diseases