My name's Gideon Hershford. I'm a hepatologist from Toronto. My colleague, Dr. Taylor from Denver, is a pediatrician. We've adjusted just the moderation so that I'll help with the moderation today. We've got four, well actually not four, five amazing talks. The purpose of this SIG-driven meeting was really to allow people to listen to experts in the field around the gut-liver axis in cholestatic liver disease. As all of you know, both for children and for adults, these are very impactful diseases and our therapies are not precise enough and our science has really taken off understanding the microbiome, understanding bile acids and new opportunities for treatment. So that was the purpose in choosing the variety of different outstanding speakers today. We're going to have 15-minute talks approximately and we can do some discussion at the very end. With that, I'll ask Dr. Taylor to introduce our first talk. We're just going to flip it around for some technology reasons. Our first speaker will be Dr. Mark Sundren, who's going to be presenting on the liver-gut axis and speaking about bile acid-induced polarization of intestinal T-lymphocytes. So I see that the setup here is specifically for those of us that are right-handed. Unfortunately, I'm left-handed, but I'll do my best. Thanks for the introduction. Good morning, everybody. I'm really excited to talk to you guys today about a really important, but I think still emerging area of bile acid research, and that's how bile acids interact with gut immune cells generally and T-cells specifically. For today, I'm going to focus on what we know about what happens to T-cells in the intestine, but I know later on in the session we'll hear about bile acid-dependent regulation in other tissues, including the liver. So I have a couple disclosures. Let's see. There we go. Okay, so I have a couple disclosures, but what I wanted to start by doing was just sort of place the emphasis on CD4-positive so-called T-helper cells, right? So my lab has been really interested in understanding how CD4 T-cells develop and function for many, many years, and largely the interest is based on the fact that we all know that protective immune responses to intracellular pathogens like viruses, extracellular bacteria, fungal organisms, and even extracellular parasites like Helminthic worms are all different, and it's really the CD4 T-cells that tailor those immune responses. I'm not going to go through all the specifics. This is basic immunology 101, but essentially I'd just like to remind you guys that there are two subsets of CD4-positive T-cells that develop in the thymus in parallel, so FOXP3-negative naive cells, FOXP3-positive Tregs. Those leave the thymus, enter peripheral circulation, and it's really the job of the naive T-cell to mount these protective inflammatory responses against pathogens. It's the Tregs' job to suppress T-cell responses against autoimmune autoantigens. So if a naive T-cell sees a peptide antigen and is not suppressed by Tregs and also gets requisite co-stimulatory signals, it becomes activated. It starts making all these cytokines and growth factors like IL-2. Those activated T-cells, importantly, become really locked in peripheral lymph nodes, where they spend several days sort of sensing and responding to other cytokines that are present in the microenvironment. And so depending on those types of cytokines that usually come from innate immune cells, including antigen-presenting cells, that will specify which type of effector or regulatory T-cell emerges. And so the Th1, Th2, Th17 cells, of course, those are all pro-inflammatory, and then you can also get induction of peripherally-induced Tregs. But the point is here is that that initial polarization step doesn't actually affect the immune response, right? So it's only unless and until those cells leave the draining lymph nodes, migrate into non-lymphoid tissues, and then re-encounter antigen, that's where they secrete their polarized sets of cytokines that instruct the rest of the immune response. But the other important point about this slide is that you can see that most of the regulatory action actually happens in the lymph node. And so for a long time, a central dogma of T-cell immunology was really that the responses that we see in peripheral tissues are sort of a static byproduct of all the regulation that happened days before in the lymph node. But this was really flipped on its head starting about 10 to 15 years ago from work by Diane Mathis, Christophe Benoit, and others, in which Diane and Christophe's group really beautifully showed that if you isolate FOXP3-positive Tregs from different non-lymphoid tissues, those Tregs are actually more transcriptionally different than they are similar. And furthermore, they showed that those tissue-specific Treg functions played really important roles in maintaining normal tissue physiology and suppression of organ-specific autoimmunity. And so this, as a field, we became really excited about this because this now moved our thinking from treating chronic inflammatory and autoimmune diseases by systemically suppressing the immune system to potentially being able to target specific inflammatory responses in tissues. So we became interested in this, particularly in the gut in principle, because we know that the gut is one of the most dynamic and sort of unique microenvironments in the human body, where infiltrating and resident immune cells are going to be exposed to a variety of cytokines, growth factors, parenchymal cells, et cetera, that aren't present anywhere else. And so the questions that we wanted to start thinking about was, is this notion that Tregs get specialized in tissues, is that the exception or is that the rule? In other words, do all T-cell lineages become specialized in the intestine or in other tissues? If so, if multiple lineages do become specialized in the gut, how does that work? Is there a role of bile acids? Of course, we were initially thinking a lot about the role of the gut microbiome and microbial metabolites. And ultimately, how do these specialized T-cell functions in the intestine influence mucosal immunity or inflammation such as an inflammatory bowel disease? So to start getting at this question now, this was many, many years ago before single-cell analysis. So the way that we did this experiment was we essentially fax-purified what are so-called effector memory CD4 positive T-cells that are marked by the expression of CD45RO, which is a memory marker, and lack expression of the lymphoid homing receptor CCR7. And this was really important because these effector memory cells, as you can see in that fax panel, accumulate in peripheral non-lymphoid tissues such as the ileum. And so if you don't fax sort those, then you're essentially comparing transcriptional profiles of effector memory cells from tissue against mostly central memory or naive cells in the blood. And so we fax-purified these cells from Crohn's disease patients, either from peripheral blood or from the ileum. And again, the key point here is that these are the same cells, just different zip codes. And then we analyzed them by microarray. And so consistent with sort of established notions at the time that Crohn's disease and inflammatory bowel disease involves the activation of TH17 cells, we saw that a lot of the genes that were increased in T-cells, in these effector memory cells from the ileum versus blood, included a number of cytokine and cytokine receptors that are consistent with the activation of TH17 cells. Whereas in black, you can see that the transcription factors that mark different lineages, including FOXP3, TBAT, ROR gamma T, and GATA3, those were all the same. So again, suggesting that we're not just comparing TH17 cells in the ileum versus TH1 cells in the blood, for example. But what really caught our eye was all of these different genes that had no previously known immunological function and that were very highly upregulated in effector memory cells from the ileum. And so one of those genes was this gene ABCB1B, which encodes one of the multi-drug transporters that is known for spitting out chemotherapeutic drugs from tumor cells. But this, that notion that this is a simple drug pump, sort of goes against what we know about evolutionary biology because there are plenty of MDR1 orthologs in all sorts of lower organisms, and so we were really interested in understanding its physiological function. So we went on to confirm by RNA-seq analysis that if you compared the expression of MDR1 in effector CD4 T cells from the blood ileum or colon of IBD patients, you could see that it was really a unique feature of cells that are in the ileum. And we could also use a MDR1-dependent dye efflux experiment in peripheral blood CD4 positive T cells, and we saw increased expression of MDR1 as these cells acquired the expression of both integrins and chemokine receptors that are necessary to drive lymphocyte homing into the small intestine. So this was really interesting that these cells were uniquely upregulating MDR1 in the ileum, but we wanted to know what its function was, of course, so for that we had to turn back to the mouse. And we initially made an endogenous MDR1 reporter allele in which anytime a cell upregulates expression of MDR1, it fluoresces orange because it's expressing this fluorescence in transgene ametrine. And so what we found was that just like in human cells, in the mouse, these CD4 positive effector cells really uniquely upregulated MDR1 in the small intestine and specifically, again, in the ileum. And I don't have time to show you all the details, this has been published, but I'll just tell you that what we were surprised to see was that MDR1 expression in ileal T cells really occurred in a lineage-independent manner, so any T cell that migrated into the ileum turned up MDR1 expression. It appeared to be locally inducible, so cells in peripheral circulation did not express MDR1, but then when they get into the ileum, they turn it up. And then also interestingly, it seemed that as soon as they left the tissue, they turned it back off. So they need it in the ileum, they don't need it elsewhere. So why is that? And then what was potentially most surprising to us was that we figured that MDR1 would be a response maybe to effluxing xenobiotic metabolites made by gut bacteria. This was not the case because if you looked in germ-free mice, you still saw induction of MDR1 expression in ileal T cells. So what we went on to show is essentially that what the transporter is doing is it's protecting T cells from biolacid-induced oxidative stress and inflammation. So if you knock out this transporter, the T cells go haywire specifically in the ileum, they cause ileitis in multiple different mouse models, which you can then reverse or suppress by blocking biolacid absorption into the ileum. And so we still don't know exactly what the molecular function of MDR1 is. Of course, the obvious thought would be that it's a biolacid efflux transporter. We don't have any direct evidence of that. We actually think that it may be more generally involved in suppressing oxidative stress in redox metabolism, but that we're still working on that. But I think that the big thing about this paper to us was that this was really the first description that biolacids could directly have a profound influence on the biology of T cells. And so we became super excited about this, and this led to the next question to us was, how are T cells really sensing biolacids in the ileum? And so the idea here was that certainly MDR1 is not the only thing that these lymphocytes are upregulating locally to deal with biolacid-induced stress. So this notion that there may be some sort of a biolacid response pathway intrigued us. So I think that the obvious idea here would be that T cells migrating into the ileum and sensing biolacids activated some number of nuclear receptors to be able to induce these local transcriptional responses, including MDR1 upregulation. But when we sort of thought through all the possible different nuclear receptors that could be involved, the number starts increasing, right? It's not one or two things. So instead of going through sort of stepwise and analyzing one nuclear receptor at a time, what we decided to do was a pooled in vivo RDI screen in which you can knock down the expression of all 49 mouse nuclear receptors in a pool of lymphocytes and then ask which ones of those contribute to MDR1 upregulation in intestinal T cells. So the way that this generally works is that you express multiple SHRNAs against each of the nuclear receptors. You then sort and pool those cells. You then inject those cells into mice. And then in our case, the question was essentially which SHRNAs accumulate in T cells that migrate into the intestine but fail to turn on the expression of the MDR1 transporter. And shown down here in the bottom right, you can see the distribution of different SHRNAs grouped by gene in cells that migrate into the intestine and either do or do not turn on MDR1 expression. And so all of those colored bars on the left side of that graph are the SHRNAs that were overabundant in the cells that migrated into the intestine but did not turn on MDR1. So suggesting that the targets that you're knocking down there are involved in upregulating MDR1. And so there were multiple nuclear receptors. Again, none of these had known immunological functions. But we really became interested in NR1I3, which encodes for the constitutive androstein receptor. And the reason, of course, why we became interested in this is because CAR is known for promoting phase one and phase two drug and bile acid metabolism in hepatocytes, but again has no known immunological function. And CAR is normally sequestered in the cytoplasm, but then when hepatocytes become exposed to bile acids or other hydrophobic xenobiotics, it becomes dephosphorylated and undergoes nuclear translocation, pairs with RXR, and then drives drug detoxifying enzymes in transporters. And we had known from work from David Moore's group that if you knock out CAR in mice, then those mice are exquisitely sensitive to drug and bile acid-induced liver toxicity. So we were wondering if CAR was doing the same thing in T cells. And again, trying to summarize about seven years' worth of work, what we ended up showing was that when T cells migrate into the ileum, they sense the bile acids that are being absorbed via ASBT expression on ileal enterocytes. And those cells really upregulate CAR expression through a mechanism that we still don't understand how, but the upregulation of CAR in these mucosal T cells in the ileum drives their development into another type of regulatory phenotype, which is a FOXP3-IL-10 positive phenotype known as type 1 regulatory cells or TR1 cells. And again, we showed that these TR1 cells expressing CAR use CAR to promote both the expression of drug detoxifying or bile acid detoxifying enzymes in transporters, including MDR1, but also that CAR was very important in promoting IL-10 expression. And that if you knock out either CAR or IL-10 in these ileal T cells, what you end up with is an accumulation of pro-inflammatory TH17 cells and ileitis, which again can be reversed by reducing bile acid absorption. More recent work from Ron Evans' group at Salk has shown that innate lymphoid cells, which are very similar to CD4 positive T cells but lack an antigen receptor, express FXR, and that FXR activation in innate lymphoid cells in the ileum can directly suppress IL-17 expression by promoting the ROR gamma T inhibitory nuclear receptor reverb alpha. And then I also want to point out that recently within the last four to five years or so, a number of groups have now shown that secondary bile acids made by gut bacterial metabolism are also super important in the colon. And really it's lithocholic acid and a series of lithocholic acid metabolites that both induce the production of FOXP3 or stabilize the expression of FOXP3 in Tregs. Lithocholic acid activates IL-10 in mature colonic Tregs via the vitamin D receptor. And then other lithocholic acid metabolites, including 3-oxo and iso-LCA, have been shown to directly bind and inhibit ROR gamma T dependent TH17 cell function. So again, I guess at the end of this, what I want to try to summarize is how there are really unique sets of bile acids signaling through different sets of nuclear receptors to induce sort of immune tolerance in both the small and the large intestine. And I think where the field is going with this is now we really need to start more quantitatively understanding what endogenous bile acid pools really look like in the intestinal mucosa. We've gotten really good at analyzing bile acid pools in the lumen of the intestine, but again we want to know what's in the ileum or what's in the mucosa and what can therefore interact with immune cells. In order to do that, or once we do that, then we can finally ask how do those bile acid pools that are associated with intestinal mucosa change in settings of health or disease? Think about all the many comparisons that you hear about when we use sequencing approaches to be able to compare gut microbiomes. We need to do the same thing with bile acids. And then finally, the real question is how do these bile acid pools, not just individual species, interact with immune cells and modulate host immune signaling? And so there's a lot of interest in the IBD field because there's a number of reports suggesting that bile acid metabolism is dramatically perturbed in inflammatory bowel disease, but we really have to understand how those pools influence host immune responses and inflammation. And so with that, I want to wrap up and acknowledge the folks that made this work possible, including the funding sources, and thank you. I mean, that was just an amazing opening. Thank you very much. You've got another ten years' work and we'll bring you back. So we're now going to flip back to the start of the program, and we're going to have a talk from Dr. O'Neill from North Carolina. Dysbiosis as Pathogenic Factor in Cholestatic Liver Disease. Can we turn down the lights up there? It's very difficult to see the screen. That's a good question. Can we adjust the lights? We're going to try. Perfect. Thank you. Can you help with the lights as well? Can you help with the lights? Thanks. Thank you very much. Good morning, everybody. Thank you to the organizers for allowing me to give this opportunity to give this talk. Before I start, and it's quite fitting, we started with an IBD immunology talk because we have one of the grandfathers of that, and that's one of my mentors, Balfour Sartor, of which a lot of my microbial and notobiotic work has been done under his tutelage. That I have no relevant disclosures. And so I'm a transplant hepatologist at the Cleveland Clinic, and so I would be remiss not to talk about the clinical side before getting into the nitty gritty. And whenever I think about a disease, it hearkens the faces that I see. And this is a face that I first saw growing up in Chicago, probably the greatest running back, arguably the greatest running back ever. Sweetness, for those who are young, this is Walter Payton. He also had PSC and died from complications of it. And with that, talking briefly about PSC, it's a multifocal, biliary, pro-inflammatory disease with progression of strictures and in some, a progression into parenchymal fibrosis and ultimately cirrhosis. Strong links to IBD and GI and hepatobiliary malignancies. Treatment landscape is bleak right now, but hopefully there's light. Currently, there's no cure with notable transplant despite post-transplant recurrence. And so what I want to just focus on and what we've been focusing on with the great talks yesterday and continuing on today is understanding the pathogenesis and the role the microbiota plays in potential therapeutic potential. Potential therapeutics. And with that, one of the hearkening plausible theory of PSC pathogenesis and its relationship to colitis is involved in altered gut microflora, producing potentially toxic or immunostimulating byproducts that translocate into the hepatobiliary system leading to inflammation, injury, and progression of disease. And with what's been known over the last 10 years is that we do have notable pathobionts like Enterobacteriaceae E. coli, Klebsiella pneumonia, E. faecalis, VLNL, and the predominance of E. faecalis is even seen in the biliary system. And there's a decrease, there's two stories to this. It's not just the pathogenic bacteria. We also got to hearken to protective bacteria of which a family of bacteria called Lactosporaceae have been shown to at least be associated with protective effects in disease models. And hearkening back to the importance of the microbiota, so despite colectomy before liver transplant, there's been association of recurrent PSC. And I wanted to, in one of these seminal papers that really focus on and demonstrate transferability of disease in a mouse model, it was by Nakamoto back in 2019 in which he inoculated germ-free mice with stool from PSC UC patients with association of TH17 priming, but with DDC replicated disease manifestations in the liver. And they were able to isolate three bacteria, Klebsiella pneumoniae, Proteus mirabilis, as well as Enterococcus gallinarium, of which the function of is this increasing permeability by forming these unique pores. Leading to a hypothesis again of a functional balance of resident intestinal bacteria facilitating hepatobiliary inflammation and fibrosis by production of both protective and detrimental byproducts of which I will show you examples of now. But first I want to talk about the mouse model in which I used. For those that are in the field, this is the prevailing PSC model. It's the MDR-2, not the MDR-1 knockout mouse. But its role is known to be phosphatidylcholine floppase deletion of which leads to pericholangitis and periductal fibrosis triggered largely by increased permeability in the biliary epithelium demonstrated here. But caveat to this model is that it doesn't develop colitis nor does it develop glandular carcinoma. So what's the overall role of the microbiota in this PSC model? And with that, when we made it germ-free represented in gray here and brown representing the SPF for conventionally raised mouse, what you notice here in this Kaplan-Meier curve is a fatal phenotype by at least seven and a half, median time point of about seven and a half weeks that can be rescued by transfer of stool from SPF mice and rescue at least 75 percent of them. The rescue also leads to functional decrease in cholestasis represented there. Furthermore, the mechanism of this particular facet is in germ-free animals, there's a shift in total bile acid, hepatic bile acid pools of which we talked about agnosium yesterday of how toxic that can be in this mouse model. And so we hearken to block this with the ASBT inhibitor. By blocking it in the terminal ileum, you reduce reuptake of bile acids. Noted in pink are the germ-free mice that were blocked that we used the ASBT inhibitor. We demonstrated with increased fecal bile acids compared to the control. And with that led to, again, decrease in total bilirubin, which is a marker of cholestasis, as well as total serum, total bile acids. In summary, demonstrating that the overall microbiota, at least in this particular model, leads to essential protection that we believe is related to bile acids and likely other signaling byproducts. So we utilize antibiotic, selective antibiotic treatment to try to isolate which groups of bacteria may be driving the disease. And here shows histologic inflammation score, CK19, to look at ductal proliferation as well as fibrosis. And what we notice here, it's vancomycin-sensitive populations, the depletion of which leads to worsening disease, both inflammation as well as in fibrosis, suggesting this is the protected population, of which we did a number of studies. And then we're able to find candidate microbes, Clostridaceae, as well as Lachnospiraceae, which are exquisitely vancomycin-sensitive relative to neomycin, which really targets gram-negative and metronidazole for targeting mostly anaerobes. And furthermore, when we treated these MDR2 animals with antibiotics, we saw, again, reduction in, and then we pleaded them with a 21-strain consortia of Lachnospiraceae. We saw reductions in histologic liver inflammation, ductal proliferation, as well as reduction in fibrosis, suggesting protection. And we narrowed down on its role on fibrosis further by trying to understand what they produce. And Lachnospiraceae are one of the major producers of short-chain fatty acids, which play a number of roles, including inducing colonic Tregs and maintaining tolerance. And what we saw here is, in this model where we utilized vancomycin to really suppress any vancomycin-sensitive populations, including Lachnospiraceae, and we gave exogenous short-chain fatty acids, a result of which led to reduction of fibrosis node and bridging fibrosis up here, and improvement after that. And this is only after two weeks. And we also saw changes in collagen fibrosis gene expression. Furthermore, we also then selected by mass spec'd the high-producing versus low-producing short-chain fatty acid Lachnospiraceae, and were able to replicate the data in where we saw histologic decrease in fibrosis, but there was no change in inflammation, suggesting the other 18 species that we didn't isolate played a role in inflammation. Furthermore, this multifaceted bacterial group were able to also modulate more pathogenic species, of which we were able to isolate translocans in the liver, which were E. faecalis and E. coli. And in this particular model, when we treated them, when we looked at liver E. faecalis after antibiotic pretreatment and Lachnospiraceae complementation, we saw a reduction in E. faecalis, and it was selective as another pathobiont, Klebsiella pneumoniae, was not suppressed. So it's a very targeted role. And we also were able to show that Lachnospiraceae also suppressed colonization of E. coli in this antibiotic pretreatment model. Furthermore, we took these isolates of E. coli and E. faecalis and gave it back to our germ-free mice, of which rapidly induced cholestasis, noted here with an increase of serum total bile acids compared to the germ-free MDR-2 control at the earlier stage, which they aren't as sick, but in the later stages they are sick. So you see an accelerated cholestasis, and also we also see it's reflective in total bilirubin. Furthermore, inoculating them with the germ-free mice with just E. coli and E. faecalis leads to liver-related mortality, which is accelerated and protected when we treat them with Lachnospiraceae plus the pathobionts relative to control. So in summary, to this part, we believe the Lachnospiraceae at least are one of the key bacteria that really targets a particular pathobionts that may have a protective role in the future. So the key takeaways is animal models are vital for elucidating the mechanistic roles of microbial agents in PSC. Resident microbes exhibit a protective function within this model, and Lachnospiraceae play diverse and distinct hepatoprotective roles, notably underrepresented in PSC patient populations. Thank you. Thank you very much. We'll now move on to our next speaker, Alexander Mifke, who will be presenting on intestinal bile acid transporters and receptors as therapeutic targets in experimental cholestasis. I would like to thank everyone for attending this SIG program, and I would like to thank the previous speakers for setting up and building sort of a little bit momentum here. And actually, your guys' work has really inspired me to do studies we are showing here today, and I'm glad. I guess I have to. So my talk will primarily focus on two aspects of the gut-liver axis. In the first part, I would like to show you some recent results from our group on investigating how FXR agonists actually work and whether it makes a difference whether you activate FXR in the gut or in the liver or in both. And in the second part, I would like to present you results which show us that the intestinal bile acid transporter plays an important role in mediating effects of the gut-liver axis as they relate to liver immune responses and also to the disease severity, especially the sex dimorphism we find in cholestatic liver disease. So we know that FXR agonists already have been trialed in clinical trials for PSC, and they have different properties. Some FXR agonists particularly activate FXR in the gut and others in the gut and the liver, and so we used the MDR-2 knockout mouse model, which was introduced previously, and tested two different FXR agonists, one which has a systemic FXR activation and the other one which is intestinal restricted FXR agonist, and we investigated in a short-term experiment, only seven days of treatment, how these affected the bile acetomyostasis in sclerosing cholangitis and how they affected the sclerosing cholangitis phenotype. And as you can see, both FXR agonists, the systemic in red and the intestinal restricted one in blue, they both very efficiently reduced bile acid de novo synthesis in the liver as measured by plasma C4 levels as well as bile acid concentrations in the liver. But only the systemic one prevented progression of the disease. Only the systemic one decreased bile duct proliferation. Only the systemic one decreased ALT and alkaline phosphatase levels in the blood. And only the systemic one was associated with a decrease in pro-inflammatory cytokine production, IL-1 beta, in the liver. That raised the question of whether there are other FXR-expressing targets in the liver besides hepatocytes. And then, of course, you land on hepatic macrophages as one of the cell population-mediating inflammation. And in culture, these hepatic macrophages do express FXR, and most importantly, when we expose them to LPS and TCDCA, those hepatic macrophages upregulate and produce IL-1 beta, which is decreased in dose-dependent fashion in presence of an FXR agonist. And those effects were not seen in macrophages from FXR knockout mice. Coming back to an in vivo situation, we compared two different mouse genotypes, wild-type mice, and we compared them to FXR transgenic mice in which we deleted FXR in myeloid cells with a lisincre driver. We challenged those mice for seven days with DDC to induce sclerosing cholangitis, and for six days out of these seven, we treated them as an FXR agonist. And you can see in the liver, we see a similar reduction in bilycid synthesis and bilycid concentration, and we see a response of the liver to FXR agonist in wild-type mice and in transgenic mice, shown here with a SHIP induction in this liver. They do reduce bilycid synthesis, bilycid concentration, but only wild-type mice respond to these FXR agonists by reducing IL-1 beta, and most importantly, only the wild-type mice show a reduction of the sclerosing cholangitis phenotype in response to FXR agonists, measured here with ALT, alkaline phosphatase, and bilirubin levels in the blood. So in summary, at least in our sclerosing cholangitis model, only systemic FXR activation is associated with reduction in the sclerosing cholangitis phenotype, but with a protection from progression of sclerosing cholangitis, and we showed in our investigations that FXR not only controlled bilycid production in the hepatocytes, but it also controlled the response of macrophages to intrinsic and extrinsic exogenous danger signals. It controlled the production of IL-1 beta, and I have not shown you here that this IL-1 beta is very important in licensing Th17 cells, licensing CD4 and CD8 cells, and producing IL-17 and interferon gamma, and when we treat these mice with FXR agonists, with systemic FXR agonists, we not only have a reduction in bilycid synthesis, but we also have a reduction in pro-inflammatory cytokine production and licensing of these T cells. So the next line of work was directly inspired by Mark's paper from Immunity 2017, showing the responses of T cells to bilycids. So we asked the question whether regulatory T cells in the liver are responsive to bilycids. So we asked what happens during cholestasis with these regulatory T cells. We used the DDC mouse model. We challenged them for two weeks with DDC, and then we switched them over to regular chow, and you can see during DDC challenge, there's a progression in sclerosing cholangitis. There's a rapid increase in hepatic bilycid concentration in the left, and there is a rapid increase in ART, alkaline phosphatase, and bilirubin levels, which all go back to normal when we switch them to regular chow. And importantly, during the time of severe cholestasis, regulatory T cells measured by flow cytometry contract in this hepatic lymphocyte population. So we asked the question of whether bilycids directly reduce regulatory T cell gene expression. We measured, we purified Tregs and we cultured them for two days in presence of either TCA or TCDCA, and we can see that TCA reduces FOXP3 expression in these regulatory T cells by 50%. And when we co-cultured regulatory T cells pre-treated with TCDCA, those pre-treated regulatory T cells had a decreased capacity in suppressing CD8 proliferation. So TCDCA not only represses the master transcription factor for regulatory T cells, it also reduces its function. Taking a deeper dive, we went back to the DDC model. We did single-cell RNA-seq and single-nuclear attack-seq in these mice on parenchymal and non-parenchymal liver cells at the various time points during cholestasis and after cholestasis to look at gene expression and integrate those information with accessible chromatin to get an insight into the gene regulation. And I very much focus here on the regulatory T cells. You can see on the left are regulatory T cells in this serial experiment prior to cholestasis, and you have the typical transcription factor FOXP3 is activated and controls various genes. And then during cholestasis, FOXP3 activation is decreased and you have an activation of Th17 transcription factors, for instance, RORC, regulating and upregulating IL-17A and IL-23R expressions, typical for Th17 cells. And the question came up yesterday in the bile acid symposium, so that's why I include here an extra piece of information. How are these bile acid effects mediated and what do we have candidate by single-cell RNA-seq? We don't see any expression of FXR or TGR5 in our Tregs, but we see expression of S1PR1 and S1PR4. These two sphingosine 1 phosphate receptors may be involved in bilaset sensing. S1PR2 has been shown in macrophages to mediate effects of conjugated bilasets. And we show here on the left that knockout Tregs from knockout mice for TGL5 or FXR, they are not protected from TCDCA-induced FOXP3 expression. But when we block S1PR1 with a small molecule or S1PR4 with a small molecule, you see a dose-dependent protection from the TCDCA reduction of FOXP3 expression. So that gives us evidence that these receptors may be involved in mediating the effects of conjugated bilasets on regulatory T-sets. Coming back now to the gut-liver axis, if we target the gut-liver axis by blocking reclamation of bilasets to the liver with an IBET inhibitor, can we restore protective Treg responses in these livers? So we treated the MDR2 knockout mice with an IBET inhibitor, SC435, for two weeks. We have shown before that that treatment greatly reduces liver bilaset, serum bilaset concentration, and all indices of liver injury in this mouse model. But now here we show in FOXP3, GFP, MDR2, transgenic mice that there is a three-fold increase in regulatory T-sets upon treatment with an IBET inhibitor. And that increase is associated with a decrease in plasma cytokine levels, for instance, TNF-alpha. Next, we investigated whether these regulatory T-sets, which expand during IBET inhibitor treatment, are actually relevant in controlling injury responses. So we basically have now four groups of mice. We have MDR2 heterozygous mice as control mice. We have in blue MDR2 knockout mice untreated. We have MDR2 knockout mice treated with an IBET inhibitor and with isotopic control IgG. And then on the right, we have the MDR2 knockout mice treated with an IBET inhibitor and anti-CD25 to deplete regulatory T-sets. You can see that upon cholestasis in the control mice, there is a decrease in regulatory T-sets compared with non-cholestatic controls. IBET inhibitor treatment increases the Tregs, and CD25 depletion nicely depletes the regulatory T-cell compartment. And when we look at just bile acid concentration treatment with SC4-SB5, whether we deplete the Tregs or not has a great reduction in liver bile acids. But when we look at liver disease phenotype, only mice treated with SC4-SB5 and intact regulatory T-sets showed a reduction in ALT and alkaline phosphatase levels. Treg depletion made those mice resistant to the protective effect of IBET inhibitors. And that was also true for the liver histology. Fibrosis was only reduced in mice treated with SC4-SB5 and intact regulatory T-sets. That was true for the other features of sclerosing cholangitis, peripartal inflammation, ductal proliferation, necrosis, and fibrosis. So in summary, TC-DCA represses FOXP3 and Treg suppressor function. Cholestasis induces a Th17 program in Tregs, and TC-DCA effects are mediated by S1P receptors 1 and 4. And IBET inhibition restores protective hepatic Treg responses in this mouse model. So the last part was inspired by studies showing that there seems to be a female predominance in MDR3 disease, ABCB4 mutation. There was a nice study published in Gastroenterology in 2008 where they looked at 32 patients who presented with unexplained cholestatic liver disease. They did sequencing, and 11 out of the 32 had a heterozygous mutation in ABCB4, and 8 out of the 11 patients were all females. So that raised the question, is there a female predominance for ABCB4-mediated disease? And obviously, we work with the MDR3, MDR2 knockout mice, so it made sense to look at this, and others have described this, of course, before. There is indeed in the knockout mice a dramatic difference between male and female mice. The female mice have much advanced in age-matched mice liver inflammation, fibrosis, ART, alkaline phosphatase liver, and bilirubin levels are all higher in female MDR2 knockout mice compared with age-matched male. And this advanced phenotype is associated with increased bilacid concentration in the female mice in the liver, but then surprisingly, with increased ASBT expression in the females, leading to less fecal bilacid excretion. So these mice are worse off, and they are still promoting reuptake of bilacid in the gut. Is this cause or consequence? That's, of course, a question, and I don't know whether we can totally answer it, but we did a few experiments to look at this being the cause for the problem. So we treated MDR2 knockout male mice for five days with estrogen, and we can see that these mice decreased FXI expression in the gut, which was associated with an increase in ASBT expression in the gut of these male MDR2 knockout mice, and an increase in indices of liver injury, in particular ALT levels were increased upon estradiol treatment and bilirubin levels. So in order to test the hypothesis that intestinal FXR is critically important for mediating those sex differences, we looked at three different genotypes of mice, wild-type mice, and then we used global FXR knockout mice, and we used villan-quay FXR flox mice, in which we deleted FXR only in the intestine. And you can see here between these three mice, in global FXR knockout mice, we found FXR deletion in the liver. The other two strains, of course, have FXR expression there. In the gut, we have FXR deleted in the global knockout mice and in the VCF mice, and that differences in FXR expression are important for ASBT. We have sex difference in ASBT expression only in the wild-type mice, and deletion of FXR, either globally or intestinally restricted, attenuates and abrogates those sex differences in ASBT expression, and they are directly related to the fecal bile acids, the liver bile acids, and the total bile acid pool size. We have only a sex difference in fecal bile acid excretion in the wild-type mice. Liver bile acids in this model in mice when they are challenged with 0.1% DDC for seven days, we see only a sex difference in liver bile acids in the wild-type mice, not in FXR, and in particular, not in VCF mice. And that is directly related to the sex difference in the sclerosing cholangitis phenotype. Only wild-type mice show the sex difference, not the transgenic mice. And that is also directly related to the liver histology. We have advanced fibrosis only in wild-type female mice, and advanced ductal proliferation. Summarizing that part, at least in our experimental model, estrogen has an important role on IPED expressions through FXR in the gut, which increases bile acid pool size, predisposes to cholestatic injury, and I have not shown you that. We have evidence that it also increases its sensitivity to IPED inhibitor therapy. So bringing this all home here, experimentally we show that FXR agonist therapy requires FXR activation in hepatic macrophages. Cholestatic liver and microenvironment shapes Treg responses, and that may be important for effects of anticholestatic therapy on hepatic immune function. And that estrogen, there is an estrogen-intestinal FXR-IPED axis, which underlies in part the sex dimorphism we observe in this experimental model, and would be interesting to see whether that plays out. I was asked whether this picture relates to my lab. Unfortunately not. In number, but the people who come to our Pediatric Liver Disease Symposia, they could all work in the lab, and they would advance the field. So we have another, we have the seventh Pediatric Autoimmune Liver Disease Conference in Cincinnati in April, and please show up. Thank you. So that was another fantastic talk, which really challenges this division between, you know, bile acids and immunology. So it's my great pleasure to invite Dr. Allegretti from the Brigham to come up to the podium and to talk to us, given her own personal research experience on treatments targeting the intestinal microbiome, antibiotics, prebiotics, and FMT. Thank you, Dr. Allegretti. Thank you everyone for having me. My biggest disclosure is that I'm not a hepatologist. I'm the medical director of the Crohn's and Colitis Center here in Boston at the Brigham and Women's Hospital, and I'm also the director of our fecal microbiota transplant program. So today I've been tasked with really discussing how we're thinking about microbiome therapeutics and manipulation of the microbiome, specifically in PSC. And so I think when we're talking about the microbiome and microbiome therapeutics, it's really important that we are all speaking the same language. And so I always like to start with some basic definition and concepts. And so when we discuss the microbiota, this is really the collection of microbial species that form a microbial community. The microbiome is the collection of genetic material present in the genomes of the microorganisms present in a community. And so I think we often use those terms interchangeably, but they are different. Dysbiosis refers to a derangement in the microbiota of a community, and a commensal is a microorganism that lives in intimate contact with another, deriving benefit without harming. And so in health, we think about the fact that really there's not just bacteria living in and around us. We've got viruses, fungi, archaea. But when we think specifically about the bacteria, there are really four dominant phyla, and particularly the firmicutes and the bacteroidetes make up the bulk of a healthy gut microbiome. And so if we think about the basics of how this works, the abundant intestinal flora is tolerated through complex host-immune interactions, and disruptions to this balance has been implicated in the pathogenesis of several infectious and inflammatory GI diseases. So what could possibly go wrong? We know that a dysbiosis disrupts health. There have been several dysbiosis-related diseases, inflammatory bowel disease being one of them. And we know that there are several therapeutic disruptions, including antibiotics. And now we are sort of looking at these therapeutics to see how we can manipulate the microbiome to the patient's benefit. And so specifically as it relates to PSC, this is sort of my obligatory overview of PSC. I don't need to tell this audience. But we know that this is an inflammatory and fibrotic destruction of bile ducts and will progress to cirrhosis and often require liver transplantation. And many of these patients, especially in the West, have concurrent inflammatory bowel disease. And unfortunately, there still is no effective medical therapy. And so the problem here is that dysbiosis can lead to inflammation, impairment of the intestinal epithelial barrier, and immune dysregulation. The portal vein transports products to the gut microbiota directly from- excuse me, of the gut microbiota directly to the liver, which is felt to potentially contribute to the duct pathology seen in PSC. And human studies have consistently demonstrated an altered gut microbial composition in PSC characterized- I don't know why this is moving on its own, but- characterized by reduced colonic alpha diversity and beta diversity, as well as shifts in multiple bacterial taxa. And so what is the solution to this problem? Well, we think about restoration of healthy intestinal flora through manipulation of the gut microbiome, which has gained a lot of interest in the treatment of GI diseases. And manipulation of the gut microbiome- I don't know why it's doing that, I apologize. Manipulation of the gut microbiota holds promise as an effective therapeutic strategy in PSC. And so I'm obviously going to focus on FMT, but there are several ways that you can manipulate the gut microbiota, including antibiotics, pre and probiotics, fecal microbiota transplantation, and more recently we're seeing some promise with phage therapy. And these are among the most commonly utilized way to restore gut microbial composition and functionality. And so I will just briefly touch on antibiotics. I know vancomycin in PSC is a hot topic, and clinical trials have shown that daily antibiotic regimen can improve liver function, blood biomarkers in PSC. Vancomycin has been by far the most widely studied microbiome targeted agent as a therapy for PSC. Oral vancomycin exhibits a low side effect profile and has shown promising results in two randomized control trials, demonstrating reductions in both ALKFOS and Mayo risk scores. However, the ideal dose, the ideal formulation, and duration of treatment for oral vancomycin still remains unknown. Other antibiotics such as metronidazole, rifaximin, minocyclin, and azithromycin have demonstrated variable and less impressive clinical results, and these studies are unfortunately small and underpowered. With regards to prebiotics and probiotics, prebiotics are non-digestible compounds thought to promote growth or activity of beneficial microbiota, which may have benefit in other liver diseases, but unfortunately no studies to date have investigated the role of prebiotics in PSC. Probiotics, we have one randomized placebo controlled crossover trial investigating a probiotic containing four lactobacillus and two bifidobacillus species in 14 patients with both PSC and IBD. And in this trial, no improvement in ALKFOS, other liver biomarkers, or clinical symptoms were appreciated. And so that leads us to fecal microbiota transplantation. So what is this? This is the installation of minimally manipulated microbial communities from the stool of a healthy donor into a patient's GI tract. And this is how FMT is really distinguished from defined consortia or probiotics because it's not just bacteria. It's viruses, fungi, metabolites, everything that comes with whole stool. And this really lends to the degree of complexity of this therapeutic. And so the FDA still considers this to be both a drug as well as a biologic or tissue, so it is doubly regulated by the FDA. In this country, this is not, nor will it ever be FDA approved, unfortunately. The FDA continues to maintain their policy of enforcement discretion, stating that you can use FMT to treat patients with C. diff, not responding to standard therapy without an investigational new drug license. This is an ever evolving area. You still need to state that it's investigational in your informed consent process and discuss the real and theoretical risks sort of per this FDA policy. This has been evolving. In 2016, the FDA stated that they would likely enforce an IND requirement for anyone using material from a stool bank. Many of us actually went to the FDA in 2019 to try to fight this requirement. And then I think we all know what happened shortly after that. There was a global pandemic and the group in the FDA that regulates FMT also regulates vaccines. And so this got put on the back burner for a while and they sort of resurfaced back in the end of 2022 and actually did enforce this IND requirement. So if you are using material from a stool bank, there is an IND requirement now. The onus right now seems to fall on the stool bank and not the practitioner. But this remains a very challenging space, especially to do clinical trials because of the regulation that the FDA has put on this therapy. When we think about the safety of this, you know, I have been doing this for almost 15 years. I believe in the safety of this therapy, but there have been some safety concerns that have been brought forth in the literature. And when we think about MDRO transmission, the FDA in the summer of 2019 had recently reported that there were two immunocompromised patients that developed a systemic infection with an extended spectrum beta-lactamase, an ESB-L E. coli, after FMT. And unfortunately, one of the patients did die from this infection. When we sort of look at the details of these cases, we note that these were not done for C. diff. These were under IND protocols, one for hepatic encephalopathy and the other for graft-versus-host disease, so very immunocompromised patients. And the donors had not been tested for ESB-L previously, even though this was being done in an IND-approved protocol. And so scenarios such as this really do emphasize the importance of meticulous attention to donor testing for potential pathogens and really the need for standardization. The pandemic affected this space as it did with everything in medicine, and what happened with FMT is that initially, when the pandemic started, material that was produced after December 1st, 2019, so left the donor after 2019, was not eligible for use per the FDA. Everything had to go into quarantine. OpenBiome, which is the largest stool bank in this country, immediately implemented swabbing of the donors, but the FDA really did insist on stool testing. We know that this virus is expressed in the stool, and so stool testing needed to be developed and validated, and that is essentially what took the whole of 2020 to do. And so now all stool aliquots are also tested for the virus. No reports of transmission of SARS-CoV-2 have been found post-FMT to date. There have also been other infections that have been reported post-FMT, EPEC and STEC, other strains of E. coli. Specifically, we have always screened for E. hec and STEC using an enzyme immunoassay. EPEC has not traditionally been screened because it's not felt to be a pathogen. However, in the beginning of 2020, there were four cases of STEC, two cases of EPEC that were reported post-FMT. They all resulted in self-limiting diarrhea. However, because of this, the FDA now mandates sort of the broad PCR testing of all donor aliquots. With that being said, though, FMT has a very well-established role for the prevention of recurrent C. diff. It is ID and GI C. diff guideline approved in both the U.S. and Europe, and this is across several different delivery modalities. But obviously, my involvement in this space in both IBD and C. diff really led us to think about where this type of therapy may also have potential benefit. And so our group started by trying to understand sort of the microbial pathogenesis in PSC a bit better. This was an early study that we did looking at the bacterial composition of bile in patients with PSC compared to non-PSC patients all presenting for outpatient ERCP. These patients were essentially matched based on pre-antibiotic use, so there isn't a skew of antibiotic use just in the PSC cohort. And what we did was we actually sampled from the entire GI tract all the way down through the biliary tree. And when we specifically look at the bile composition, this is from culture, we see that in the non-PSC patients, you really see what looks like gut flora. But in the PSC population, we really see a much less broad diversity of organisms and really a trend towards strep viridans or no growth at all. When we actually sequenced the bile, we see that there is a lot more diversity again in the bile of non-PSC patients compared to those with PSC. And so after this, this really led us to think about the use of sort of microbial manipulation to manage the bile of non-PSC patients. And so this led us to think about FMT in this space, and the hypothesis was that FMT will correct a dysbiosis that has led to the hepatobiliary inflammation seen in PSC and will therefore improve LFTs and hopefully slow progression to cirrhosis. And so this is not a new trial but still unfortunately the only FMT trial in PSC to date. This was an open-label pilot study of 10 patients with PSC and co-occurring inflammatory bowel disease. All patients received a single FMT via colonoscopy, all from the same donor. We collected blood and stool prior to FMT and we followed patients through week 24. Blood was tested for LFTs throughout as well as inflammatory markers. And we did stool microbiome assessments throughout as well as bile acid profiling. And so to be enrolled in this study, patients had to have a confirmed diagnosis of PSC with concurrent inflammatory bowel disease. They had to have LFTs that were at least 1.5 times the upper limit of normal. Patients were not allowed to be on ERSO in this study so we actually did wash patients out of ERSO for a minimum of four weeks prior to the screening visit. And we assessed the primary endpoint as a decrease in alkaline phosphatase by 50% or greater at any time point through the study. We also of course looked at microbial diversity. And so again we enrolled 10 patients in this study. Most of them were men. You can see the mean baseline alkaline phosphatase was 489. Most of these patients, nine had ulcerative colitis and one had Crohn's colitis. None of these patients were on biologics or other advanced therapies for their inflammatory bowel disease. At the time I think the question around safety of FMT using biologics was still unknown when we designed this study. So all the patients were on either nothing, mesalamine or azathioprine. So with regards to the outcomes, we really saw no relevant adverse events. There was one adverse event that was felt to be unrelated to the FMT which was a sinusitis that resulted in a hospitalization. And then we did have one patient who really did not tolerate being off the ERSO and ultimately had to be restarted and withdrawn from the study. So with regards to the clinical results, three of the 10 patients did achieve the primary endpoint of a 50% decrease in alkaline phosphatase by week 24. Seven of the 10 patients experienced at least a 30% decrease in one of the other liver biomarkers by week 24 as well. We also note that the FMT did shift the taxa composition towards that of the donor in all of the patients with PSC. We see a nice increase in alpha diversity as early as week one and we did follow these patients out through week 24 and we see that that was actually sustained even with just a single FMT. And so we also assessed engraftor strains. If you're not familiar with the term, engraftor strains are those that are present in the donor, missing in the patient pre-FMT and then present in the patient at one week post-FMT. And when we looked at the engraftor strains, we see that they actually represented a diverse set of taxonomic classes so it wasn't just one main class of bacteria. And so many possible strains could be deriving the therapeutic benefit and it's felt that potentially benefit may also be derived not just from one strain but synergistic combination of strains. And so when we looked at the presence of engraftor strains and also the improvement in alkaline phosphatase, we see that the more engraftor strains present, the more dramatic the improvement in the alkaline phosphatase was. And so I think we were all very excited when we saw the results of this study, recognizing that it was a small proof-of-concept study. And I think many people have asked me when the next study is coming. And as I mentioned, I think the regulation of FMT and doing clinical trials in this space has been quite challenging, especially through the pandemic. However, this has been a very exciting year for those of us in this space. We now have two FDA-approved LBPs, live biotherapeutic products, that were approved for the use of prevention of C. diff. And I think now there is a renewed interest in really studying these therapies as a way to manipulate the microbiome in diseases such as PSC. So I think in the very near future, we're going to have some new data for you on some of these new products in both inflammatory bowel disease and PSC. I think the world of traditional FMT studies is probably coming to an end, unfortunately. So with regards to phage therapy, if you're not familiar, bacteriophages are self-replicating viruses that infect bacteria with high host specificity. Lytic phages attach to bacterial cells and inject their viral genome into the host. The phage genes exploit the host gene expression machinery, leading to the replication, synthesis, and assembly of phage progenies. And finally, the bacterial cell is lysed by phage products, thus releasing the phage progenies into the environment. And so I will just end by mentioning the study that was presented by a Japanese cohort earlier this year. And so this study looked at bacteriophage therapy against pathologic Klebsiella pneumonia, which ameliorated the course of PSC. They had previously looked at the composition of the microbiome in PSC patients and found that Klebsiella pneumonia, Proteus, Mirabalis, and Enterococcus galanearium were noted to be present as significantly in this cohort. And this trial investigated the clinical applications of bacteriophages against specifically the Klebsiella pneumonia isolated from a patient with PSC. The Klebsiella pneumonia was selected based on the pathogenicity of intestinal damage, bacterial translocation, and susceptibility to hepatobiliary injuries in a mouse model. And they found that oral administration of a phage cocktail significantly reduced the Klebsiella pneumonia levels in fecal samples and subsequently attenuated hepatobiliary injuries. So in summary, manipulation of the microbiome remains an area of therapeutic interest in PSC. FMT is safe in this population. Improvement in overall microbial diversity persists in this population. Abundance of engraft or OTUs present in patients with post-FMT correlated with a decrease in ALKFOS. And further studies certainly are needed to determine the utility of isolating engraft or OTUs and to understand the role of the microbiome in the pathophysiology of PSC. And I think now we see phage therapy is also showing promise. So thank you so much, and I look forward to questions after. Wonderful. I'd now like to introduce our final speaker, Michael Tronner, who will be presenting on therapies targeting bio-acid transport and signaling. Thank you very much, respected chairs, dear colleagues and friends. I want to thank the organizers for inviting me. And my slides are up already, okay. Okay, so these are my disclosures. I was asked to give a kind of a clinical wrap-up to where we are standing, and I really want to focus on three aspects. FXR ligands, IPED and ASPD inhibitors, and norethrodeoxycholic acid, recently named as norecholic acid or NCA. So this slide is to remind us where we stand with our therapies within the enterohepatic circulation of bio-acids beyond ERSA, which Alan Hoffman correctly has labeled as the first enterohepatic bio-acid therapy. So bio-acids, while they circulate through the enterohepatic circulation, signal through FXR and TTR5, and this signaling function is amplified by gut hormones such as FGF19 and GLP-1. And when we talk about FXR ligands, and we heard about that in the talk from Alexander, we, on the one hand, can focus on FXR ligands, which are more restricted to the gut or more systemic FXR ligands, which also primarily hit the liver. Then, in addition, we have recombinant FGF19 as an FXR downstream target. And we also can interfere within transport, within the enterohepatic circulation with ASPD inhibitors, which we've heard about already. Resins, of course, also have the same mechanism. NTCP blockers. And also modification of the bile salt export pump may be a therapeutic avenue. In addition, bio-acids, as we've heard, are interfering with the gut microbiota, and this is giving rise to a whole new range of bio-acids. Mark has nicely referred to, you know, the atypical iso-, allo-, and oxo-bile acids, which have these exciting immunomodulatory functions and may be restricted to certain compartments in the intestine. And we also have, for example, polyhydroxylated bile acids, which form in B-Cep knockout mice, protecting them through their anti-inflammatory properties in contrast to humans who develop these severe P-Fig phenotypes. And then we have norocolic acid, a drug undergoing cholehepatic sunting, so a principle outside the enterohepatic circulation. And in the interest of time, I will focus on three aspects. I will not be able to talk about, you know, the correctors and potentator modulators of transport function or siRNA gene therapy or mRNA strategies, which are very exciting developments in this field. So let's start where we stand with FXR ligands. And as you are aware, this already has entered clinical reality in the treatment of PBC, where obeticholic acid is a first-approved FXR ligand has become the second-line therapy. And now we have these other options also with PIPA ligands and also butazenide as second-line therapy. In addition to biochemical improvements, I think it's important to emphasize that these drugs have been shown to also have potential long-term benefit. For obeticholic acid, that has been demonstrated for a synthetic external cohort and for beta-fibrate here, the Japanese data on liver transplantation-free survival in a retrospective study. So why do these nuclear receptor-targeted therapies work so well? And why are, for example, biological approaches in PBC and also in PEC so disappointing? And I think the interesting thing is that these nuclear receptor-targeted therapies are immunometabolic drugs, targeting both biliary homeostasis and inflammation. And we also should remind ourselves that with bile acids for FXR, we've heard about fatty acids, which are ligands for PIPA. There's a lot of biology behind that, that these nuclear receptors are really in the center of interest. And we've heard actually from Alexander, his beautiful presentation, pointing out that this balance in terms of modifying biliary homeostasis and immunological inflammatory processes may differ profoundly between systemic and intestinally-restricted FXR ligands, which both repress bile acid synthesis, as we've seen, but are profoundly different in repressing inflammation, which was normally seen with systemic FXR ligands, shown here in red. I would like to point out that the studies in MDO2 knockout mice are very difficult to interpret, in my mind. We have strain differences, we have microbiota differences between laboratories. So in FUBN mice, we could demonstrate that Xylofexo, which is this gut-restricted FXR ligand, actually was able to attenuate liver fibrosis and also improve liver biochemistry. And I would like to point out that the concept of gut-restricted FXR ligands developed in an area where we wanted to avoid systemic side effects, such as pruritus and dyslipidemia. So that was the concept behind targeting the gut and, of course, targeting the suppression of bile acid synthesis. And we've also heard yesterday in the post-credit course that mice have a much higher rate of bile acid synthesis than humans. So maybe animal data, which target bile acid synthesis, are kind of predestined to have a less effective approach, for example, in humans. And I think maybe this is what we've seen with the PREMI study, with the Phase III study with Xylofexo, which I've presented at the ISLA-ILC meeting, which was terminated prematurely for futility reasons and did not show an impact on the primary endpoint, which was the one-stage increase in liver fibrosis. So maybe, Alexander, you were right in a way. And unfortunately, we don't have the samples to really investigate the differences in the engulfment of the immune response, for example, in these studies. But I think this would be extremely interesting. I would like to point out that FXR and bile acid signaling, of course, when you go to the gut, also has a central role in regulating gut integrity. You know, not only regulating gut microbiota, but also the mucin defense, the tight junction, also the gut vascular barrier, and, of course, also immune function. And there is preclinical data that obeticolic acid, the non-steroidal FXR agonist, is attenuating DSS or TNPS-induced colitis, for example, an effect which was lost in the FXR knockout mice. And I would like to submit that FXR is a very interesting target, especially in PSC, where you have the link to inflammatory bowel disease, which may drive the disease and actually targeting both the liver and the gut, maybe one-stop shopping for the liver and the gut. And I also would like to point out that in current clinical trials with PSC, we actually excluded patients with severe IBD, so there was no way that this signal would be detected. So I would not entirely give up about targeting FXR, also by testing restricted FXR ligands in a disease such as in PSC. Let's move to IBD inhibitors. And here we really already have entered the clinical space. As you are aware, two IBD inhibitors are now approved for the treatment of pruritus in Aller-Schill's and in the PFIX, and the principle is that by reducing the return of bile acids in cholestatic injury, there is reduced bile acid accumulation. And the first generation of drugs is actually those luminally restricted IBD inhibitors. And this really landmark, iconic trial in Aller-Schill's with maralixibat and in the PFIX with odivixibat, both trials actually showed that the reduction in serum bile acid levels nicely correlated with an improvement in pruritus. And also here we are seeing that this has an impact on clinical outcomes such as that the reduction of bile acid levels is associated with improvement in event-free survival and also native liver survival. So also here it's more than symptom control. It has an impact on the outcome of these patients. What about the adult space? Here a different IBD inhibitor, linorixibat, has been studied in PPC with the 2B study, which just came out now. A little bit less effective in the control of pruritus and also more side effects, especially at the higher dose, up to 25%. So there seems to be a profound difference in my mind in the biological response between adults and kids when it comes to bile acid retention and also when it comes to tolerability and GI side effects. So I think this needs some attention as well. In addition, also preclinical studies in the MDO2 knockout mouse from our lab and also from Alexander Mietke's lab have shown that both IBD inhibitors but also resins improve sclerosing cholangitis. So also the biliary phenotype. And with resins, for example, one of these mechanisms is the induction of GLP-1 in the colon. And GLP-1, in addition to the metabolic effect, also has immunometabolic and the apoptotic mechanisms in cholangiocytes, which we have not talked about yet. So this is also an interesting avenue how a metabolic gut signal may impact on cholangiocyte apoptosis. When we look at IBD inhibition, we've heard the beautiful demonstration from Alexander about the immunomodulation, but we should not forget that actually quite simple things are also happening here. For example, when you reduce the bile acid return to the liver, you just simply reduce the bile acid load in bile and promote the beneficial ratio of bicarbonate to bile acids and the phospholipid-bile-acid ratio by inhibiting IBD. So also interfere with the toxicity of bile. And when we talk about IBD inhibitors, I think we not only have to think about the ileal inhibition, but also systemic inhibition of IBD. And there are very nice data from the IBD knockout mice and also from systemic ASPD inhibitors that this accentuates the loss of bile acids via the urine. And we've heard about that yesterday from Paul Dawson that the real conservation of bile acids in cholestasis is something very important to interact. And also Paul has shown those nice data which were generated together with Jan Hengstler and Ahmed Galab in Duisburg, actually showing that also cholemic nephropathy, which I think is something very important in advanced chronic liver disease, can be attenuated by reducing the bile acid uptake, which is killing the proximal tubules in the kidney with the systemic ASPD inhibitors. And taking this from the kidney to the cholangiocytes, we also have to think about inhibiting ASPD at the level of cholangiocytes. There's very beautiful data from the lab of Heather Francis in Indiana University showing that this is interfering with the FXR-NAS cell axis and cholepathic shunting of bile acids driving the cholangiopathy. And if you take this further to NTCP blockers, there's also preclinical data in cholestasis, again in the DDC model, for example, that pulivertide, the hepatitis delta drug, actually, by interfering with bile acid uptake, may interfere with bile acid-driven inflammation. So finally, what about nor-ursodioxycholic acid? Nor-ursodioxycholic acid is a side-chain, shortened derivative of ursodioxycholic acid discovered by Alan Hoffman, which is undergoing this cholepathic shunting. So it's an unconjugated bile acid which is generating this bicarbonate-rich choleresis, and this, in the MDR-2 knockout mouse model, led to dramatic improvement, basically total reversal of the phenotype. In the meantime, we know that many things happen with promotion of bile acid detoxification, anti-inflammatory, anti-fibrotic effects, and this has led to the clinical development of NCA, which now is a Phase III trial being completed in the next year. And in addition, NCA, or nor-ursodioxycholic acid, also has profound impact on T-cell function. Keep in mind that an unconjugated bile acid does not require a transporter to access cells such as T-cells, which normally would not have a transport system for conjugated bile acids. And we could demonstrate that in CD8 T-cells, nor-urcolic acid is actually suppressing mTOR signaling and interfering with the immunometabolism, also suppressing glycolysis and switching the cells to fatty acid oxidation and reducing the proliferation of CD8 T-cells. In addition, recently also in CD4 T-cells, we could demonstrate that nor-urcolic acid is impacting on the breakdown of glutamine, on glutamine analysis, and these Th17 cell proliferation is very much dependent on glutamine, and again here this feeds to the inhibition of mTOR signaling. And in addition to reducing the proliferation of Th17 cells, we saw an expansion of regulatory T-cells, including different tissues such as the liver and the colon. I'm showing you here some data because we think that the Th17 Treg imbalance is very important both for driving PSC and the underlying inflammatory bowel disease. And for example, if you look in the CD4 T-cell transfer colitis model, we observed a profound impact of NCA here on the macroscopic images, for example, the colonic length, and also on the histology here in the CD4 T-cell transfer model where the colonic histology was normalized by NCA. If you look here, for example, at a globlet cell and mucin stainings, a nice restoration of the colon. We also tested that in a second model, in the anti-CD3 model, which results in a massive activation of a Th17 response with the reduction of regulatory T cells. And in this model, we saw a profound impact on the colonic morphology of these mice, and the repression of Th17, and again here, expression of regulatory T cells here in the colon. So it's interesting that also in human Th17 cells from patients with PSC-IBD, we saw this reduction of proliferation and also the inhibition of mTOR signaling, nearly down to a level which we observe with rapamycin. So we close here, but also here we have the theme that targeting the gut in addition to targeting the bile duct may be interesting. As I mentioned, the Phase III study will be read out next year. Of course, this study was not designed to study the impact on colitis, but I think this is something not to forget about, and NCA is also in clinical development for PPC, mainly the restoration of the bicarbonate umbrella. So I would like to close that FXR ligands have become clinical reality, OCA being the second-line therapy in PPC. IBID inhibitors, and perhaps more interestingly, ASPD inhibitors, of course, are very effective in controlling symptoms, such as pruritus in a la Shilla and PFIX. The role in PPC and PSC is currently evolving. I think those are interesting drugs, especially the systemic ASPD inhibitors. In addition to NCA, there is an ongoing study in PSC and also in PPC, and I think all of these drugs may have dual mechanisms of action in the hepatobiliary system and in the gut, which hold promise by controlling inflammation in both compartments, and perhaps also by improving gut inflammation, for example, impacting on diseases such as PSC. With this, I really want to thank my team, in particular Claudia Fuchs and Ashley Schuh and Emina Halilbasic. Here in this photo is also Gustav Baumgartner, who unfortunately passed away a few weeks ago, shortly before his 90th birthday, so Gustav Baumgartner, after his stellar career in Munich, continued 10 years in Vienna as honorary professor to support our group, and we are very grateful to his mentorship. And also our cooperation partners, we've lost Hans-Ulrich Marschall, who was really a stellar star in the bile acid field and will be dearly missed. Thank you very much for your attention. That was fantastic and a really fantastic session. We do actually have the SIG business meeting, which follows on from this meeting in this room, so we actually have, since I'm chairing that meeting, the opportunity to allow the session to run on a little bit and do some questions and answers, so I suggest if the speakers do want to come up, at least if anyone wants to ask for five or ten minutes, some questions, I think we should allow that. If people do want to take the chance, you've just heard five stellar talks from world experts, I suggest we definitely take the opportunities. I don't know who was first, so I can just start on the left-hand side. Thank you. I'm Serena Longhi from Beth Israel Harvard and I have two questions, one for speaker one and one for speaker three, if possible. Great talks, thank you for providing this. I have a question for speaker one related to the upregulation of MDR1 that you have found in CD4 effector cells once they reach the ileum. You said that is a transient expression. Back probably in 2015, there was a paper from Yale Group talking about transdifferentiation of Th17 cells going through the ileum and upregulating TR1 cell markers. I wanted to know whether this MDR1 transient expression along with your CAR expression could somehow explain this transdifferentiation of effector cells trafficking to the ileum and then acquiring TR1 cells. And then I wanted also to know whether this upregulation of MDR1 is not something that may also help immunoregulatory immunometabolites to be flipped out and therefore probably diminishing the possibility of cells to acquire regulatory properties. Yeah, thanks for the questions. So I mean, the short answer is that we have a pretty unsophisticated understanding of all of the functions of what MDR1 is doing in these lymphocytes. And it really starts with trying to better understand what it's actually flipping out of cells and or organelles. We just don't know. And a big question that we have is whether it really is participating in transporting bile acids versus different types of metabolites. We have evidence, for example, that the MDR1 transporter is really important immunologically in other cells and in other tissues even, right, that are not exposed to bile acids where it still has cytoprotective and sort of metabolic activities. And then, yeah, in terms of the stability and transdifferentiation, I mean, those are really interesting questions. And, you know, all we have right now to really understand the function of MDR1 in immune cells are just germline knockouts. And that is just not a sophisticated enough tool you're going to have to develop, right, lineage-specific drivers to be able to inducibly ablate MDR1 at different points to be able to understand what it's doing when. And the other question for Speaker 3. I was really interested in your data related to estrogen. And I wanted to understand how do you think to translate into humans? So basically, estrogen would have a sort of pro-inflammatory role mediated by biliary acids. But how would you translate this into humans? So PSC patients also might… I think, thank you very much for the question. I think estrogen, of course, has a plethora effect on almost everything, including T-cell homeostasis. So we did a very reduced approach and just looked and primarily compared, because we went in there with a hypothesis that the effect of estrogen of liver disease is on hepatic FXR. And I think one contribution maybe of that work is to show that intestinal FXR does respond, and that is enough to actually abrogate the sex differences. So I don't think it helps us to understand the full role of estrogen in controlling the predisposition of women to autoimmune liver disease and other diseases. And in PSC, it's obviously not… That's why I ask about PSC, because in autoimmune hepatitis, we do actually have some evidence that estrogen and the signaling might change in females compared to males, and females at different time points. So that's why I ask about the PSC, because obviously PSC is not such a female preponderant condition as perhaps a PBC or autoimmune hepatitis. There's a question over there. Very interesting conference. You never mentioned Peyer's patches, which in the old days were certainly considered important in the immune system of the digestive tract. Is it irrelevant, or doesn't anyone study it anymore? It's a great question. I mean, again, what I tried to do at the beginning was essentially say that the field has, in some ways, shifted towards understanding, once you get development of mature lymphocyte subsets into non-lymphoid tissues, then what happens after the fact, right? Because the first 60 years of really the study of immunology was focused on understanding how lymphocytes see antigen and get primed and differentiate within these lymphoid tissues, which Peyer's patches would be included of, right? So the idea is that the guys that get primed and polarized in Peyer's patches leave the gut, but then they're already imprinted with homing receptors to come back. But when they come back, they go into the lamina propria, right? And so that was—I apologize, I probably should have mentioned that specifically, but the idea was to understand what happens once those mature subsets get into these non-lymphoid tissues. They do recirculate in the rest of the body, too. From the Peyer's patches. They can, right, but I think that they're very much primed to, you know, more prevalently go back to the gut from where they were. Great. It's just great how simple immunology is, really. Yeah. Thank you. And to communicate that in 10 to 15 minutes. Exactly. Thank you very much. Yeah, my question to Dr. Jessica Allegretti, yeah, on your PSC patients. Since you've been giving the FMT for a long time, have you considered changing their diet to that of the donor? And also, can you formulate probiotic or prebiotics for such patients who are using the FMT? And the second one is, you know, there's controversy as to the best way to administer estrogen. So I want to ask the speaker who uses estrogen, how did you administer estrogen to your mice? Thank you. All right, I'll start. So great questions. And so we have certainly looked at diet and the effect of diet post-FMT, even in C. diff, you know, where sort of this field developed. And we don't really see that dietary shifts really affect engraftment, per se. Now, in C. diff, in other chronic diseases, such as inflammatory bowel disease, and I've also previously done an obesity study, we also looked at diet. And I think it's just really unclear the role that diet will play in terms of the efficacy of this therapy and sort of increasing engraftment. We saw really robust engraftment in this trial and in our others. So I think it has more to do with donor characteristics and sort of the host immune response and less about diet, at least as far as we know right now. And so with the question around probiotics or isolating, you know, single strains or a family of bacteria, that has been trialed. And we know that probiotics don't engraft in the same way that FMT does. So the probiotics you buy from the store, they don't stick around. So that's why really FMT and the efficacy behind FMT still is developing. I mean, we don't really fully understand how this therapy works completely because it's so complex. What I can tell you is that in the field of LBPs now, we have whole consortium products and more defined consortium products. So even the series product, which was just recently approved, are just Clostridial spores. And now we have Vedanta Biosciences, which is investigating the first non-donor-derived product. So it will be the first lab-based synthetic product. So I think we are moving there. But this has been so challenging that the science has not been there yet. Okay. Thank you. The estrogen? You know, I would say it's an early investigation. We were primarily interested in is it a protective effect of testosterone or is it a pathogenic effect of estrogen, which is sort of what was our leading hypothesis. Of course, if you want to explore this more deeply, you would have to ophorectomize these female mice and see whether you can abrogate the effect of estrogen. But we were struck after five days that we already saw that signal in the gut of FXR suppression, which, interestingly, when we looked at the liver, the hepatocyte, there was not a significant suppression of FXR. So with this preliminary data, we just moved on to further explore the gut-liver axis. So, I mean, my question is how did you administer it in this IP injection? Thank you very much. Okay. I suggest we just take the last four questions. Otherwise, I'll probably get into trouble. But this is really exciting. So a question there. Jim Boyer, New Haven. Fascinating talks, all of you. Dr. Sundar, we seem to be concentrating on you here this morning. In your transporter evaluation, did you find any other transporters upregulated by your T cells in the ileum? I mean, if you want to get rid of bile acids, you would upregulate, of course, OST alpha beta. What about that? Yeah. So, again, a great question. So we have looked at as many transporters as we can look at. And we really haven't seen, I'm trying to think, because we have competing mouse and human data sets. And I believe that we've, I think it's SLC03A1. SL3. 51. No, no, no. So neither OST alpha beta nor BSEP were upregulated, right? So these are not, these lymphocytes are not upregulating dedicated bile acid efflux pumps. So the way that we've generally interpreted this, both between the specific upregulation of MDR1 over other ABC transporters and the utilization of the CAR nuclear receptor as opposed to something like FXR, is that this is probably a mechanism that's used by circulating lymphocytes that when they get into a tissue where there's, you know, oxidizing agents like bile acids, it's really upregulating and using this machinery in a general sense to protect against oxidative stress. But what would be the substrates? What's that? What would be the substrates? Yeah, well, this is the million-dollar question. We're continuing to work on this. I mean, there's, you know, MDR1, like, you know, unlike MDR2, it's not a specific phosphatidylcholine floppase, but it has been shown at least in vitro to be able to flip different lipids. And so, you know, one of our favorite hypotheses is that potentially what it's transporting are oxidized or modified lipids with modified acyl chains and that that could be a way to prevent accumulation of oxidized lipids within cells. But, again, it's difficult to show. We just haven't done that yet. Fascinating work. Thank you. Move to that microphone. I have a question for Professor Turner. Great review. Thank you very much. I wonder, given the recent, obviously, good response and introduction of ibotin inhibitors in the cholestatic conditions, how would you comment on the possibility of using these medications in other chronic cholestatic conditions, such as, for example, chronic rejection or GVHD, as a temporizing measure for these patients awaiting different treatments? They are certainly very effective also in any disease conceptually, which is associated with retention of bile acids. The only limiting factor, which I really see in adult patients, are the GI side effects, which seem to be more, you know, more secretory, more promotility effects. So I think the systemic ASPD inhibitors, also targeting the kidney and also targeting the bile duct, hold a big promise there. And for adults, I think it will move into that direction. But do you see a value, a potential value, of any non-ibotin inhibitors for the similar cholestatic conditions? You mean NTCP inhibitors, for example? Yeah, for example. I mean, conceptually, I think, I mean, yes, yeah, but the clinical data are not there. And I think there's a lot of reluctance, you know, also from industry, moving in too many different indications. But, for example, for PSC, vodavixibat and maralixibat are also developed for PSC, and in PBC, it's linorixibat, for example. Okay, we'll do two last questions, then we'll do the business meeting. Ken? Ken Satchel, Cincinnati. A question for Mark. In terms of MDR-1, you mentioned the isomers of lithocholic acid, of which there are four, and all four are found in human feces. Do you have any evidence that MDR-1 can transport them? Because one of the big problems with those bilases is that they're incredibly insoluble. And, you know, they bind avidly to the fecal content and are really not present in the aqueous phase if you spin down stool. So I'm curious to know what your thoughts are with regard to that, you know, the transport for those particular bilases. Yeah, so our functional studies pretty clearly suggested that MDR-1 is seeming to protect lymphocytes from oxidative stress that's induced by more of the primary conjugated bilases. Taro-CDCA was sort of our model conjugated primary bilase. So if you titrate in taro-CDCA to CD4 T-cells, you progressively kill them. The ones that preferentially survive express MDR-1. And if you inhibit MDR-1 function, then those cells die. And they just don't survive. And so we did do biochemical experiments to look directly at whether or not MDR-1 was capable of effluxing taro-CDCA. It was not. It did not bind. It did not competitively bind to MDR-1 in place of these number of known chemotherapeutic substrates. And by contrast, if you add things like lithicolic acid, these much more hydrophobic, unconjugated bile acids with high passive permeability, that destroyed lymphocytes independent of MDR-1. That was just extremely toxic. So we think if anything MDR-1 is protecting cells against these conjugated primary bile acids that are generally less cytotoxic but at high enough concentrations, MDR-1 can have an effect. Yeah, if I may just ask a general question to everyone. But, you know, given the effects of toroquino that Alex showed, the anti-inflammatory effects, has anyone tried taurine supplementation? Because if you give people taurine, you shift the bile acid pool to become predominantly taurine conjugated. The human bile is predominantly glycolate and glycoquino. So I'm just, I mean, this was done in cystic fibrosis, I don't know, 20, 30 years ago. I'm just wondering, has anyone tried that in PSC to give supplementation? It might be an idea. I'm not sure this is a good idea, Ken. There's a lot of literature that an increase of taurine conjugation due to milk products, for example, gave rise to the increase in Crohn's disease and the bile filler about swastika is also linked to that. So I think there's a certain danger signal there. Well, thanks for that. And you can buy taur-ursa deoxycholic acid. You can buy it through the, and that does actually work in PBC. Yeah, but you can buy taurine. One last question, then we'll move on to the business meeting. This is brief. Cooper Adelaide. Another dysbiotic cholestatic state is total parenteral nutrition. Are there any lessons which can be learned from TPN cholestasis? That's a great question. Probably at the end of the table. I think, you know, removing the soybean lipids from TPN formulations, which was an FXR antagonist inhibiting BCEP function, has changed the field. And, of course, there's also a lot of rationale for FXR activation. But I think the clinical problem was the inhibition by toxic lipids, which were FXR antagonists. However, you still get cholestasis despite changing to omega-1 or SMOF. So there seems to be something recently about some sort of medium chain triglyceride formation in the New England Journal of Medicine. And there's very nice data, you know, on the role of FXR and FXR insufficiency in the gut in those patients. So maybe that's a role where FXR agonists maybe have a benefit in the gut. That's great. Thank you very much, everyone. We're fantastic speakers. More questions than answers.

gut-liver axis

cholestatic liver disease

FXR activation

bile acid synthesis

inflammation

microbiome manipulation

PSC

estrogen-related FXR signaling

sex differences

MDR1

tauro-CDCA

FMT

ibotenic inhibitors

ASPD inhibitors