Good afternoon from New Jersey. My name is Grace Guo. I'm the moderator for today's webinar, sponsored by MASH-LD SIG. So, today's webinar is about emerging topics in understanding mechanisms of MASH-LD pathogenesis for drug development. So, with this webinar, we hope to bring you the contribution and the mechanisms of immune cells and organ cell crosstalk in MASH development, leading to therapeutic targets. The first aim of this webinar is we would like to bring you the mechanisms of MASH development. And the second, we'd like to bring some insights for future therapeutic targets in the treatment of MASH to improve steatosis, inhibit inflammation, and regression of fibrosis. We also would like to encourage collaboration among the hepatology community group, as well as to introduce state-of-art approaches in conducting mechanistic and translational MASH research. My co-moderator is Dr. Nian He, who's on emergency transplant surgery and cannot join us today. But I would like to introduce three of our speakers, Dr. Javier Ravelo from University of Minnesota, Dr. Anna Medeo from Duke University, and then Dr. Scott Freeman from Mount Sinai at New York. So, the first speaker is Dr. Javier, who is an associate professor at the Department of Integrative Biology and Physiology at the University of Minnesota since 2018. So, in 2021, he was awarded the Keystone Symposium Fellowship. And also in 2022, he's awarded the McKnight Land Grant Professorship at the University of Minnesota. So, his research has been focused on the role of immune system and inflammation in the pathophysiology of cardio-metabolic disease, including MASH LD and heart failure. And he has published recently on the lipid-associated macrophages, mediating the beneficial effect of bariatric surgery against MASH. As well as two years ago, he published on microbiota-driven activation of intrahepatic B-cell, which aggravates MASH development through innate and adaptive signaling. Without further ado, I'm going to pass the podium to Dr. Ravelo. Thank you, Grace, for that kind introduction. So, today I'm going to try to be brief and give you the highlights, showing you some work we've recently done looking at how B-cells and lipid-associated macrophages promote the development of metabolically-associated fatty liver disease. As you know, MAFLD is a progressive condition that begins with the simple lipid accumulation, also known as osteoporosis. And through mechanisms that we don't fully understand, this benign fatty liver disease becomes an inflammatory condition known as metabolic dysfunction-associated stelehepatitis, or MASH. And so from here, things get much worse. Some patients develop cirrhosis and even liver cancer. And the transition between the simple fatty liver disease and the inflammatory condition is, as you can imagine, a key step in the natural progression of MAFLD. We don't fully understand the mechanisms driving this progression, but we think inflammation is one of the key mechanisms triggering the development of MASH. And so this is a simplified view of all the immune cells that interact in the liver leading to the progression of MASH. You have your monocytes that arise from the bone marrow. Those monocytes enter the inflamed tissue where they become mature macrophages. And recent work has shown that those macrophages can be inflammatory, your typical monocyte-derived macrophages, but there's also a new subset of lipid-associated macrophages that are involved in the processing of lipids. There's resident macrophages, also known as Kuffer cells. These cells are thought about, they initiate the inflammatory response. That's their known role. These cells die during MASH progression through mechanisms that we don't fully understand. So a lot of attention on the monocytes, macrophages, Kuffer cells, and then you have the other innate and adaptive immune cells participating in this process, including neutrophils, B cells, T cells, cytotoxic T cells, and dendritic cells. Notice how I put a question mark next to the B cells because little is known about the function of these adaptive immune cells in the pathogenesis of MASH, and that's one of the focus in my lab. So normally when you think of B cells, you can imagine these cells being important in the response to pathogens through the production of antibodies, right? That's their main function, but sometimes we forget that B cells are very important regulators of inflammation, and they do that through the secretion of cytokines. So B cells, including B cells in the liver secrete lots of TNF, lots of interleukin-10, interleukin-6, and through this cytokine release, they can communicate with other immune cells, including the macrophages, and have a very important role in the inflammatory process. And so we came across an interesting finding when I was a postdoc in Toronto. This is about 10 years ago. We had some access to liver tissue from patients with NASH, and in some of the stains, we were able to detect that B cells accumulate in these cell lumps in the patients with severe NASH. So we had a pathologist assist us, and we were able to determine a positive correlation between NASH score, so the severity of disease, and the accumulation of B cells, in this case, determined by the expression of CD19. So as NASH score increases, we have increased B cells both in the portal areas as well as in the lobular areas. And that's been shown already. It was no surprise to us that B cells increase in NASH, but what is the function of those cells in the pathogenesis of this liver disease? To study that, to answer that question, we use our mouse model of diet-induced NASH. In our lab, we use either a normal chow diet to keep the mouse healthy, or we induce NASH using a Western-type diet, a diet that is high in fat and high in carbohydrate. So the first thing we did is we assessed the frequency of B cells, and we also took a look at the subsets of B cells that are present in the NASH liver. And what we found was that the majority of the B cells were the B2 B cells that increase in the liver of mice with NASH, and these B2 cells are the classical B cells, inflammatory cells that make a lot of cytokines and make substantial antibodies. We also detected some small frequencies of B1A and B1B B cells. These cells are more rare, and they have specialized functions, including the secretion of natural antibodies. But very small frequencies, the majority of the cells were the classical inflammatory cells. Now, we wanted to study these B cells in depth, and the first thing we did is we assessed how much cytokines these B cells make in response to different stimuli when we take them out of the liver. And what we found was that regardless of how you stimulate the B cells ex vivo, B cells from a NASH mouse always make more TNF and more IL-6. So somehow these cells are wired to respond to a stimuli by secreting increased levels of pro-inflammatory cytokines. To prove causality, to know whether this response of the B cells is a bystander response, or if indeed there's a role for these B cells in disease development, we use a mouse model that lacks the heavy chain for the UMT gene. So these mice are unable to form immunoglobulin M, and so they're unable to produce mature B cells. They're lacking B cell systemic. And the most dramatic phenotype of these mice was that despite modest decreases in lipid accumulation, these mice were completely protected from fibrosis. And with that, we also detected decreased levels of ALT and AST following 15 weeks of high-fat diet, high-carbohydrate feeding. So B cells not only become activated and secrete cytokines, they seem to have a key role in MASH development, specifically fibrosis. So that was interesting to us. We wanted to know more about these B cells, and of course we used single-cell RNA sequencing to better understand the function and phenotype of these cells. And we sequenced all immune cell types in the livers of lean, healthy mice and in the immune cells of mice with MASH. We found four different clusters of B cells here in this square. Of course, we detected macrophages, T cells. They always change, but in this study, we focus on the B cell subsets. And we found that the major subset of B cells resemble that B2 mature inflammatory subset. And when we looked closely at the gene expression in that cluster, we found indeed strong signature of inflammatory factors abregulated in the Western diet-fed mice. One of the top factors was MyD88, and this is an adaptive protein that sits below the toll-like receptors and transduces all those signals coming from toll-like receptor 479 to inflammation. So this made sense to us, and to understand the role of this molecule in the B cells during the development of MASH, we generated a B cell-specific MyD88 deficient mouse that lacks the MyD88 specifically in the B cells. We fed these mice the Western diet for 15 weeks and then looked at their phenotype. There were no evident changes in the frequency of immune cells, so no defects in the number of B cells and other cell types, but those B cells in these mice were unable to abregulate activation factors, antigen presentation molecules, suggesting that MyD88 is required for the activation of these cells during the development of MASH. And in agreement with our total depletion of B cells, mice lacking MyD88 specifically in these cells were protected from fibrosis. So this is unpublished work. We continue to tackle this question of the mechanisms by which B cells become activated during the development of MASH. And so an interesting recent finding in the lab was that when we use a mouse that has a B cell receptor, that of course B cell receptors have a unique ability to recognize antigen in a highly specific manner, but if we use a mouse with a B cell receptor that has a bias towards an irrelevant antigen that is not found anywhere in nature, that mouse is also protected from some of the aspects of MASH, including fibrosis. And so this data is telling us that not only the B cells become activated, but this is some evidence that there's an antigen specific response going on during the development of a disease. So to quickly summarize our findings regarding B cells in MASH, we see evidence that B cells become activated through tolec receptor signaling and B cell receptor signaling. These cells then secrete cytokines, including IL-6, TNF, and promote fibrosis. I don't have enough time to show you, but we also see some crosstalk with CD4 and CD8 T cells, and we also have evidence that the leaking of microbial products from the gut into the liver can activate these cells during the development of MASH. So I'm going to briefly switch gears and tell you a new story that we recently published looking at how lipid-associated macrophages help the liver repair during bariatric surgery. And this is a collaboration with the Department of Surgery. We were interested in whether decreased inflammation was a mechanism by which bariatric surgery enhances the repair process following the bariatric surgery. And so the first thing we did is we assessed the macrophage content in liver specimens from people before and after the bariatric surgery. We used spatial transcriptomics to study macrophages in zones 1, 2, and 3 of the liver. We used antibodies such as CK7 and CD68 to help us annotate the zones within the liver, and then we analyzed the gene expression in these zones before and after the bariatric surgery, vertical sleeve gastrectomy. And we saw a very strong response to the bariatric surgery in the macrophage compartment. Genes like CD63, CD68, CT, SL, perilypin 2, TREM2, all of them were telling us that the bariatric surgery was dampening the macrophage response one year after these patients underwent the bariatric surgery. So to study whether that was a mechanism of bariatric surgery and not simply a response of the immune system to the beneficial effects of the surgery, we went back to the mouse. And in our lab, we have a mouse model of bariatric surgery in which we mimic what happens in humans, and we do a vertical sleeve gastrectomy. We remove about 70% of the stomach surgically, and then we place a clip in these VSG mice. The mice do well. Most of them survive the surgery, and then we study their responses either five or 10 weeks after the bariatric surgeries. We have different control groups. The main group is a sham surgery that we perfect to match the calories of the bariatric surgery. We're attempting to look at weight loss independent effects. So no surprises, the bariatric surgery was very effective at dampening NASH progression, specifically the level of triglycerides as well as inflammation. And that protection was beyond simple caloric restriction because the sham-perfect group was not as effective as the bariatric surgery group. So in the last two minutes, I'm going to just briefly show you some of the highlights. So what I'll say is that what we found is that these lipid-associated macrophages that we found in the humans are required for the bariatric surgery to exert its functions. Apologies for extending my time. If you have any further questions, we can discuss later in the Q&A section. Thank you so much. Thank you so much, Javier, for that excellent talk. We're going to leave all the questions for the final panel discussion. And then our next speaker is Dr. Anna Medeo. Dr. Anna Medeo is the Florence Michalister Distinguished Professor of Medicine at Duke University. And then she has been very active serving the NIH, AASLD, and many universities in the entire world as advisory members. For Anna May's research, for the last three decades, she has focused on liver injury and repair, particularly her research interest focused on immune system regulation of liver injury and regeneration, as well as the role of fatal morphogens such as Hatchdog pathway in regulating fibrotic response to liver damage. And her research has been funded by the NIH since 1989. Since 2001, Anna May Bail and her colleagues have been active participants in the NIH-funded MeSH clinical research network. This is a national consortium comprising eight universities to generate a national registry for patients with NAFD and also conducting multi-center clinical trials. So without further ado, Anna May. Well, thank you, Grace. I don't know how you guys can see me. Are you seeing a full screen of my slides? Yes. Okay. Well, I'm going to tell you some published data, some unpublished data that we've been working on for the last couple of years. And what I'd like to emphasize is things that we're learning about what might influence how people respond to metabolic stress. And as we all know, they've renamed NAFLD to NASLD, and that is actually a good idea because it's emphasizing the stressor that causes this kind of liver disease. So we know it's metabolic dysfunction associated liver disease, and it's a spectrum of liver damage, as you heard from Javier, where you start with a healthy liver, people get steatosis. That seems to be a pretty reversible state. You can come and go in and out of steatosis, but a subset of people who get steatosis develop steatohepatitis. Even people with steatohepatitis can improve and regress back to steatosis at normal. But once we start to see fibrosis appear, we know that people are on a different trajectory and that the fibrosis is likely to become progressive. And if it progresses all the way to cirrhosis and liver cancer, of course, there'll be liver-related morbidity and mortality. But even at earlier stages of fibrosis, we see an increase in all-cause mortality as well as liver-specific morbidity and mortality. So perhaps we can learn more about the mechanisms that are actually controlling disease progression by trying to sort of leverage clinical observations to help us focus on specific factors that we already know a lot about. And that's where I'd like to highlight the emphasis on aging, because we know that age is a risk factor both for NASH, or MASH, I guess I should say, and it's also a risk factor for cirrhosis. And so that's interesting because really this trajectory from steatohepatitis to cirrhosis is really reflecting tissue degeneration. And it's well known that aging is a risk factor for all kinds of degenerative diseases. Oops, I'm not sure I can advance it, let's just see, did that advance the slide? No? No, maybe putting in the forward button, the arrow, see if that can... Yeah, I'm trying, let's see. Uh-oh, it advanced when we tried it earlier, but now I'm not advancing, let's see, how can I get that to happen? Arrows are not advancing it, let's just see. Nominees, please, why is push the... There we go, did that advance it? Oh, perfect, yeah, there we go, okay. So here's the hypothesis, aging, which we know is a risk factor for both MASH and cirrhosis, causes liver degeneration, or at least promotes liver degeneration because it's causing old cells or senescent hepatocytes to accumulate. And what I'm showing you on the right side of this figure is a very beautiful cartoon that somebody else made and published in the Frontiers of Molecular Science in 2021. But this will sort of give you the idea that I'm going to try to look at now in metabolic liver disease. So the idea is that normal hepatocytes see a stress, and we already talked about the fact that metabolic stress is what hepatocytes are seeing in MASL. And then some of these cells actually are adapting to try to survive that stress, but maybe not fully successfully, and they incur some DNA damage, and so then they start to stop themselves from proliferating so that they can fix themselves. And if that happens long enough, they become senescent. Senescent cells are viable but vulnerable, but they remain alive. And they're actually quite metabolically active, and they make what is called a SASP, which is a secretome. And the secretome is very rich in pro-inflammatory factors and pro-fibrogenic factors. And so these senescent hepatocytes are lying there in the process probably of dying, but they're alive, and they're making things. And that's going to influence other cells in the microenvironment as well as tissues outside of the liver. And one of the things that happen is they recruit in immune cells, because they're actually trying to get themselves eliminated from the tissue, because building up senescent cells in tissue can be bad. It's bad because it promotes inflammation and fibrosis, but it's also bad because some of these senescent cells can apparently evade or escape senescence, but they've been reprogrammed. And if they undergo a stochastic malignant transformation, these are the seeds for liver cancer. So in the next several slides, I'm going to show you evidence that this actually happens in MASL. And so why do you care about this? Well, if you've been reading the aging literature, and probably even the late press, what you've seen is a trickling out, and now the floodgates are kind of opening with a lot of papers coming out and saying that aging is reversible. And it's reversible because you can clear these senescent cells using drugs called senolytics, but you can also reprogram the cells that are in the process of becoming senescent or that have already become senescent by doing various things. And I'm going to focus on the last one here for just a minute and tell you about some really interesting studies where people have hooked two young mice together, two old mice together, or a young mouse with an old mouse, and what they can actually see that when you hook the young mouse to the old mouse, the old mouse becomes younger and the young mouse becomes older. So aging is actually transmissible. So can we start to look at any of this kind of stuff in the literature? Well, we can, because the aging field has defined what they call hallmarks of aging. And there are things that you can actually look for in tissue sections or in the blood, and they're summarized in that circle that you can see. They talk about genomic instability, telomere attrition, epigenetic alterations, and we'll come back to that. But you'll see one of the hallmarks of aging is cellular senescence. And that's pretty convenient because we know that senescent cells are identified by the upregulation of cell cycle inhibitors in their nuclei, things like p16 and p21. So we can look in our sections of humans who have various stages of MASL and try to figure out are these cells accumulating, and if they are accumulating, does their level of the burden of senescent cells in the tissue correlate with disease severity? And so here we took a very nice cohort that we have assembled at Duke of about close to 400 people that were obese and had adenoma liver tests and underwent a liver biopsy to see whether they had MASL or not, and if so, how bad it was. And it turned out by the time the biopsy was done, about maybe close to 70, 80 of these patients no longer had any disease in the liver that really looked like MASL, and there are controls. And then we have close to 300 people where they had various stages of MASL. And here we can isolate the RNA from these patients and do RNA sequencing experiments and then look for pathways that are activated in the people that have NAFLD versus those that don't or across the spectrum of MASL. And in figures A and B, what you can see is this is a gene signature that somebody has reported that is a signature of senescence. And you can see in figure A that the people that have NAFLD are more likely to have that signature in their liver than the people that don't. And in figure B, you can see that the people that have more advanced fibrosis when they have MASL are more likely to have the signature than the people who have MASL with less severe fibrosis. And the graphs in figures C and D are looking at the expression of a couple of those hallmarks of aging that I told you about, CDK, these levels on the bottom are P16 and P21. And you can see that the cell cycle inhibitors are more upregulated in NAFLD than in the controls in our population. And in D, you can see within the population of people that have MASL, there's more of the cell cycle inhibitors as the disease gets more severe, as the fibrosis gets more severe. And that's just summarized in the panels on the right, where you can see we did immunostaining for P21 or P16, and you can see the very nice correlations with the severity of ballooning, the NAS score, and the fibrosis scores. So this work shows then that there is a correlation between the signature of aging, if you will, and severity of MASL. And this is interesting, because I think maybe now this will give us some new ideas about prognostic or diagnostic biomarkers. The other reason this is interesting is because not only is aging inevitable, it's actually predictable. And this slide sort of summarizes some very exciting work that maybe I certainly wasn't aware of until I got into this area, but people have discovered that the effects of aging on our DNA are so predictable that you can actually predict chronological age by looking at changes in methylation of circulating DNA, and that's called the Horvath clock. And on the bottom, where you see that line in the red and blue dots, the chronological age would be the black line, and so the older you get, the more of these marks you have, and you can predict age by looking at methylation in cell-free DNA in the blood. But what Horvath discovered is that it's not exactly, people don't exactly fall on that line. Some of them are a little bit above it, some of them are a little bit below it. And that's because aging can be modified. And if you, for example, inherit the progeria gene, your biological clock would be, your biological age would be much older than your chronological age, and you're going to die of atherosclerotic vascular disease when you're five. Epigenetics can also modify the clock, and in fact, that's a tractable way to modify it because this can be modified by some of the things that we do for NAFLD, and I'll talk more about that later. But here's a paper that Rohit Lumba and I published in JCI Insight in 2017, and we looked at this Horvath clock in the peripheral blood of a group of people who had NAFLD and varying degrees of fibrosis. And the line that goes across on the bar graphs on the right-hand side, figures B and D, would be the chronological age. And if you're above the line, that means your biological age is older than your chronological age. And if you're below the line, your biological age is younger than your chronological age. And then you can see that in figure B, the NASH patients are in yellow and the controls are in black, that most of the NASH patients are above the line. And if you look on the figure below in figure D, where you see the more fibrosis, the more orange the bars are, that most of the orange bars are above the line. So it sort of suggests that biological aging is accelerated in people who get NAFLD versus people who don't. And it's probably particularly accelerated in the people who get more advanced liver disease. And of course, this was cell-free DNA circulating around in the blood. And the question is, did that really come from the liver? So what I'm going to show you now is that, well, at least some of these cells may have been in the liver because we could treat aging in the liver by these approaches that other people have identified, like clearing the senescent cells. And if this is relevant to the disease, then getting rid of the senescent cells should improve the disease. So let me shift gears for just a minute and tell you how we're going to clear the senescent cells. So one of my colleagues at Duke, and it says in review on the slide, but actually this paper has since been published. And what he discovered was that some of the receptors for the clotting system that are present on a lot of cells in the liver, including hepatocytes, can bind different things that will stabilize senescent cells. So hepatocytes and other cells of the liver have thrombin receptors. They also express this receptor called EPCR and another receptor called PAR1. And when thrombin receptor binds thrombomodulin, it recruits activated protein C to the other receptor. And then that causes a very specific cleavage and a particular residue on PAR1 that will trigger survival signaling. And our colleague has shown that this survival signaling promotes the viability of a whole bunch of different kinds of senescent cells in various tissues. And here we were looking to see, does it also happen in the liver? So if you look on the right-hand side of the slide now, what you'll see is that in patients with NAFLD, they may have a little bit more PAR1 mRNA, but if you look below it, they have a lot more PAR1 stabilized on hepatocytes. And thrombomodulin expression also goes up in the NAFLD patients relative to the controls. And you can see that at the mRNA level, but also if you look on the immunohistochemistry, the interaction between thrombomodulin and PAR1 stabilizes the thrombomodulin and PAR1 complex and causes this continuous survival signaling. And we wanted to know whether this was really related to senescence, because our colleague had shown that it's senescent cells that do this. So here we overexpress P16 specifically in hepatocytes using antiviral vectors. And you can see that if you look in the histologic images in the middle of the slide, when we put in P16, we get senescence because everything turns beta-gal positive, which is a marker of senescence. But we also see a dramatic upregulation of the thrombomodulin and PAR1 complex. And we really did induce senescence because overexpressing P16 prevented these mice from regenerating their liver normally after a partial hepatectomy, or if we gave them carbon tetrachloride. So we now established that in the liver, when you make a hepatocyte senescent, they upregulate this system that should assure their survival and allow them to hang out in the liver. So now we want to ask, do these non-regenerative or senescent hepatocytes promote liver degeneration or cirrhosis? And so one way that they could do that is through this secretome that I told you about before. So here we can induce senescence in cells, either HOH7 cells, which is a malignant hepatocyte cell line, or in mouse hepatocytes by overexpressing P16. And then we can collect this in conditioned medium and put it on Scott's LX2 cells and see whether we activate the cells. And for some reason, the control is not showing up in the blue, but anyway, you can see little stars above all the red bars that basically what this secretome, if you will, of the senescent hepatocytes is activating the LX2 cells to become more myofibroblastic. And they may move with a lot of time. Yep. So what I'm going to show you now is that basically if we do that, we can clear these thromological and pharyngo-positive senescent cells and improve inflammation and fibrosis. And that this is probably because of factors that the cells secrete, and of course my favorite factor are hedgehog ligands, and these senescent cells make hedgehog ligands that are biologically active. And I want to end by saying that this is not pie in the sky because a lot of the therapies that we're already doing for NAPL are actually reprogramming the epigenome. And I'm going to just end by showing you some exciting work that will entice you to get on your Peloton because we know that exercise, one of the things it does is reprogram the epigenome and recent work shows that it increases the amount of taurine in your blood. This nice paper was published by another group in Science in June, where they showed that aging decreases taurine in all species that they tested, including people. And then if they can replenish taurine in the blood, that they can reverse all of these hallmarks of aging, at least in mice. So the bottom line is that mastoid cirrhosis and cancer result from liver degeneration, which is caused by the progressive erosion of the calcite functions that are controlled by genetic and epigenetic factors. This reflects a loss of epatocyte resiliency, which is biological aging. The aging causes the dysfunction. It precedes and predicts epatocyte death, deficient regeneration, and progressive organ fibrosis. Fibrosis is a biomarker of inhibited liver regeneration. It causes wounds that won't heal, cirrhosis and cancer, and it should be preventable and reversible because biological aging is preventable and reversible. And so we can talk about these questions in the session immediately thereafter, but I want to leave you with the optimistic note that immortality is achievable, at least for some jellyfish. I'll stop here and be happy to take questions when we get to the session. Thanks again. Thank you so much, Anna-Mae, for that interesting and insightful talk. We're going to leave the questions for panel discussion. I'm going to try to stop sharing here if I can. Yeah. Stop share. Did we get it? Perfect. Thank you. Yeah. Last but not least, we have Dr. Scott Freeman to give the last final talk. So we all know Dr. Freeman very well. He is the Dean for Therapeutic Discovery and the Chief of the Division and the Liver Disease at Mount Sinai. He's best known for his role in isolating and characterizing the hepatic static cells, which we all know are so important for liver fibrosis. Because of his achievement and the contribution to research, he has been awarded numerous awards, including the 2003 Falk Foundation Award in Germany, 2012 ESOS Recognition Award, and the 2014 Shanghai Magnolia Gold Award. In 2014, Dr. Freeman was also awarded the Distinguished Achievement Award from the ASLD. So for today, he's going to talk about the novel understanding of inflammation and fibrosis for the treatment of MASH. Dr. Freeman. Hi, Grace, thank you for including me. It's a pleasure and an honor to share this webinar with you and my other speakers, co-speakers. In the interest of time, I'm going to move at a quick pace and paint largely in broad strokes. These are my disclosures. We all know this spectrum. What I want to point out is that the way we have scored MASH has relied on the NAFLD activity score, which was developed in the NIDDK Clinical Research Network. And then these three features are scored separately from fibrosis. I emphasize this because while we recognize that inflammation is a critical component histologically of MASH, nonetheless, the lobular inflammation is, the approach of quantifying lobular inflammation by biopsy is too inexact. And so histologic features of inflammation in this disease are not sufficiently sensitive to help us really score. In fact, the only thing that correlates with progression of disease is fibrosis, and I'll emphasize that in a minute. A reminder that hepatocellular carcinoma is also an outcome of end-stage fatty liver or steatotic liver disease, and we need to be mindful of that because carcinoma can also develop before the patient has cirrhosis. Inflammation is also a critical component, not only of MASH, but also of the propensity to develop liver cancer. This is a landmark paper two years ago that established that patients who have hepatocellular carcinoma with underlying NAFLD have a different outcome than those who have other etiologies when they are treated with checkpoint inhibitors. What you can see in both the discovery and the validation set is the blue line indicates a poor prognosis for those patients with NAFLD treated with checkpoint inhibitors who have hepatocellular carcinoma. It suggests that the inflammatory milieu of this disease is different from other chronic liver diseases. And the authors in this paper speculated that there may be NASH-related A-baron T-cell activation that leads to impaired immune surveillance in this disease compared to viral hepatitis. Much more is coming out now, which I don't have time to review, but the real point is that the inflammatory milieu of MASH or MASL-D is quite distinct, and the standard histologic markers don't distinguish those differences. I emphasized also that fibrosis drives outcomes. This is most clearly demonstrated by this other landmark paper from Arun Sanyal, looking at the NASH Clinical Research Network outcomes over 10 years of follow-up with biopsy at the onset of that 10-year period. Patients with F3 or F4 cirrhosis in NASH had a much increased risk of death from any cause, but in particular, there was a marked increase in hepatic decompensation events as shown here, as well as an increased risk of hepatocellular carcinoma. The correlation of fibrosis with outcomes explains why increasingly clinical trial endpoints for experimental drugs must show a benefit in fibrosis if they're to affect long-term improvement in patient outcomes. As Dr. Guo mentioned, my interest has been in hepatic stellate cells and the fact that they undergo activation, that's shown here in blue, where they accumulate extracellular matrix, or SCAR, and other changes in the sinusoid also lead to dysfunction of the liver and loss of differentiated function. We also know that stellate cells themselves have a whole host of potential mediators and markers that could emerge as targets for antifibrotic therapy. This is from a review I wrote with a visiting scientist, Akuma Tsuchida, some years ago, who is still quite accurate in defining both the membrane, the cytoplasmic, as well as the nuclear changes that occur in stellate cell activation. Again, all of these represent potential therapeutic targets and many are being pursued already in clinical trials. But we know that the disease really starts with the hepatocyte, with accumulation of fat, insulin resistance. And so if we think about the drivers of disease, we look to the adipose, as well as the dysfunction of the hepatocytes through ER stress. This ignites inflammation that elicits chemokine signaling, innate immunity, and inflammasome activation. You heard about that from our first speaker. But probably most importantly, the fibrogenic drive is thought to be stimulated by the development of what's called lipotoxicity, where soluble mediators from a fat-laden hepatocyte, including free cholesterol, Indian hedgehog, and osteopontin, are secreted and lead to paracrine stimulation of activated stellate cells that make collagen. So this is the framework for how inflammation and fibrosis figure into the overall construct of the disease. There are also epigenetic changes in stellate cells. We heard a little bit about that from Dr. Diehl. To me, the most transformative technology that has arrived in the last 40 years that I've been doing research has been single-cell methodologies. And now the ability to sequence the entire transcriptome of individual cells and understand the nuanced differences and the heterogeneity of different cell types of the liver has really been transformative in understanding and dissecting signals. You heard a little bit about heterogeneity from the first speaker, also a little bit from Adam A. This is from a study in mouse using a model called the FAS-FAS model from the laboratory of Dr. Brenner and Tatyana Kisileva. And they described a number of different subsets of stellate cells. So it's not just quiescent, activated, deactivated, and senescent. There are intermediate stages of stellate cells that have unique features that speak to the increasing appreciation of their heterogeneity. At the same time, in fact, before this mouse paper appeared, a landmark paper from Prakash Ramachandran and the group at Edinburgh used single-cell sequencing for the first time to really define the different subsets of the inflammatory and fibrogenic cells that comprise the fibrotic NASH-related milieu. When fibrosis progresses, at some point cirrhosis is developed. And several years ago, we, as a group, wrote an editorial that's illustrated here that reminds us that just because a patient has F4 cirrhosis, that does not mean they are all the same. And in fact, while F1 to F3 is non-cirrhotic, once a patient develops F4 cirrhosis, they still have several years of progressive decompensation, starting with compensated, then thickening of the scars, and ultimately decompensation that leads to either need for liver transplant or death. And with these progressive changes, there's a rise in the portal pressure or hepatic venous pressure gradient. As we begin to think about treating advanced fibrosis, we have to identify what we think will be the best stage for cirrhotic therapies. And most would argue, by the time the patient is decompensated, the horse is out of the barn. And instead, we need to really think about stable cirrhosis as a potential target for antifibrotic therapy. And those therapies are likely to be different than the treatments we would apply for F1 to F3. That's illustrated in part by this study done from our laboratory by Dr. Shuang Wang, who is now on our faculty, in which, sorry, in which she used glass imaging to make the liver translucent, as you see here, and then identify stellate cells through Desmond staining. What you can see is there's a thickening network of intercellular communications or foot processes as disease advances, and that's quantified here. This tells us that the entire fibrotic milieu changes from mostly paracrine to also largely autocrine. And that's illustrated in a piece that actually preceded our work, and these were collaborators of ours, defining a new approach or understanding of fibrosis, much the way we distinguish hot and cold cancers. We can now think about cold fibrosis, where it's only stellate cells, whereas hot fibrosis also includes the macrophage, shown in blue. And that can be translated into different stages of NASH in patients. Here, you can see inflammatory NASH with a lot of inflammation representing a hot fibrosis, whereas burnt out, so-called NASH cirrhosis, where there's less inflammation, really probably represents the effects largely of autocrine simulation by stellate cells upon each other. So in essence, this cold fibrosis is fueled by autocrine signaling mediated by their physical interactions. And I encourage you to read more about this in our paper from Science Translational Medicine. Now, overlaying all this are targets that also are defined by where they intersect with the different stages of pathogenesis. There can either be drugs that affect fat and metabolism, insulin sensitization and signaling, inflammatory cells, in particular interleukin-11, as well as direct antifibrotics that target activated stellate cells. Currently, therapies that are most promising are in this group here, the drugs such as the thyroid hormone beta that interrupt normal lipid homeostasis, but also drugs that affect weight loss. Currently, there are no antifibrotics that directly affect stellate cells that look promising yet, but we expect that to change. Just to introduce one of the newer cytokines that's emerged is interleukin-11, largely based on the work from Stuart Cook in National University of Singapore, Duke, in which he and his group have identified interleukin-11 as a very pivotal cytokine that both can drive hepatocyte dysfunction, fibrosis, as well as inflammation, with promising early results suggesting that anti-interleukin-11 or interrupting interleukin-11 signaling can have a beneficial effect. Finally, to build on Dr. Diehl's comments, I want to introduce the idea that stellate cells also become senescence, and the lovely paper by Scott Lowe and his group identified UPAR, urokinase-plus-minergen activator, as a particular feature of senescent stellate cells. And then what they did is they generated CAR-T cells, shown here, that specifically depleted the liver only of UPAR-expressing cells. You can see that here, before, or in an untreated animal, there's lots of beta-galactosidase activity consistent with senescent cells. Those are completely eliminated when CAR-T cells are applied or administered that kill only UPAR-expressing senescent stellate cells. And what's striking is that during this process, albumin actually increases. So it suggests that if we clear senescent fibrogenic cells, we may be able to improve function. This approach for CAR-T therapy was taken to a higher level by work of Jonathan Epstein and Drew Weissman. Many of you must be aware that Dr. Weissman and his colleague at Penn won the Nobel Prize yesterday for developing mRNA lipid nanoparticles that are the basis of the COVID vaccines, among others. And this is an elegant approach where rather than administering CAR-T cells, one administers a lipid nanoparticle that targets these cells, and then has an mRNA that can be released and engage a fibrogenic receptor. They used the studies in heart. I encourage you to look up this paper and also a summary that I wrote based on this work in New England Journal last year. So let me just summarize. Features of inflammation and fibrosis are highly heterogeneous based on the etiology, genetics, microbiome, and systemic factors. Specific subsets of inflammatory and fibrogenic cells discovered using single-cell technologies may hold the key to unearthing novel targets for therapy. And these successful treatments will need to arrest or reverse fibrosis either indirectly by altering inflammation or directly by targeting stellate cells. And finally, emerging strategies targeting key cytokines such as interleukin-11 or using cell-based therapies such as CAR-T cells are novel emerging approaches that need validation in clinical trials in patients. So thank you again. I hope we have a couple of minutes for questions. Thank you. Thank you, Scott, for that really insightful talk. Now we open our panel probably for a five to six minutes discussion. I don't think the people who registered to the webinar can join our discussion here, but I'll start with some questions. So I'm going to start with a question from questions. So we all know like majority of the clinical trials for MESH or fibrosis has been failed and that there are some promising therapies but all coming with very severe side effects. So I want to ask the panel, what do you think in the future would be a more practical effective therapy to halt MESH as well as fibrosis? Well, I think if I can jump in, I think the first approach which looks at least partially successful is the efforts to improve the metabolic state of the liver by reducing liver fat and inflammation. That may be sufficient, but remember that only 25% of patients in the recent phase three trial by Madrigal actually had benefit. So it speaks to the fact that no one therapy is going to be perfect for every patient. And the disease is much more heterogeneous with different treatment targets among different subgroups of MESH. I would echo what Scott said, Grace, that no therapies that have only targeted the stellate cells seem to be important or that have targeted the matrix. On the other hand, any of the therapies that induce systemic benefit seem to have had some benefit on fibrosis. So even if we look at weak therapies like lifestyle modification or whatever, they have been shown to improve fibrosis. So this idea of maybe targeting the low hanging fruit is not such a bad one, right? The default position of the liver seems to be to regenerate. And so if we can maybe take the pressure off and stop asking it to multitask all the time, maybe slowly things will improve. I think the other lesson that we've learned over time is that perhaps we're kind of pressing the liver to do something faster than it normally would. And that's the lesson from bariatric surgery. Remember bariatric surgery, when we looked at the two year data, we didn't think that it really reversed fibrosis. In fact, it might've even been making fibrosis a little bit worse. But when we look at the five year data, it really suggests that there was reversal of fibrosis. And so it may just take longer for the markers that we're currently using to evaluate fibrosis to be able to pick up these changes. So maybe it's not as bleak as we think it is. We may just have to wait longer to see what that. My two cents, targeting inflammation has been underwhelming in clinical trials. And so I believe more precise therapies are needed to not simply think about preventing inflammation, but what aspects of inflammation need to be targeted specifically. Some of the inflammation as we call it could be needed, could be required for repair. So both Anna Mae and Scott mentioned targeting or reversing senescence would be a very promising target to basically treat NASH or liver cirrhosis. So would that also come with the concern that it might also affect the tumor genesis in the long run? Well, that's a very interesting question, Grace. The relationship between fibrosis and cancer is extremely complicated. And there are studies on both sides of the argument saying that fibrosis accelerates cancer or fibrosis may encapsulate and protect against cancer. I think most of the evidence suggests it promotes cancer, both through physical interactions with tumor cells through the stiffness of the matrix, as well as creating a unique inflammatory milieu that involves not only checkpoint inhibitor exclusion, but also DDR2 signaling. So I think this remains one of the critical issues because it's possible that we will stop NASH progression, but we won't delay the emergence of cancer. We need to be mindful of that possibility. And I want to just emphasize the point that, all cells can become senescent. And we have a paper that we published showing that actually senescent stellate cells are less fibrogenic, not more fibrogenic. So you have to be careful which approach that you use. Sometimes getting rid of senescent cells might be bad, but I think we don't understand the nuances of senescence well enough yet to be able to target that more specifically. I agree. Yeah, I think it may be that senescent cells do something else, but they aren't the most fibrogenic. They may be the most pro-carcinogenic. They may be the most pro-inflammatory, but we're still trying to sort this out. Now that we have signal cell techniques, I think we'll really make progress. All right. Thank you so much. I think it's perfect on time. Now it's one o'clock. And we'd like to really thank the three panelists so much for giving this updated, as well as very interesting and insightful talks. And I hope see you all at ASLD in November. Awesome, thanks. See you in Boston. Bye. Bye, bye guys. Thank you.

MASH-LD

pathogenesis

mechanisms of inflammation

mechanisms of fibrosis

therapeutic targets

immune cells

stellate cells

B cells

lipid-associated macrophages

senescent cells