Good afternoon. My name is Natalia Nieto, and on behalf of my co-chair, Lisa Fang, I would like to welcome you to the Liver Cell Biology Special Interest Group. This year, the program will focus on the gut-liver interaction in liver disease. This program has been organized by the steering committee made of Steve Weidman, Paul Monga, Udayanapte, Yasuko Iwakiri, Judy Popov, Wenxin Ding, Takeshi Saito, and Petra Hirsova. We will have five speakers, followed by a 10-minute period of live question and answer. The first speaker will be talking about FXR-mediated intestine-liver communication, and the talk will be given by Grace Huo, who is professor in the Department of Pathology and Toxicology at the Ernest Mario School of Pharmacy at Rutgers University. Good afternoon, everybody. Thank you for the organizers, and thank you for the audience for this year's ASLB meeting. I'm really delighted to give this talk to all the audience and colleagues here. For today's talk, we're going to touch base on bioassays and also tissue-specific FXR functions, and then focus on the role of FGF1519 in regulating liver functions. Finally, we're going to discuss the pros and cons targeting FXR and the bioassay pathway to treat liver diseases. The research has shown that bioassay synthesis in mice and humans are different. Specifically, in humans, we have two pathways, classical and alternative pathway to synthesize folic acid and chenodeoxycholic acid. This will be conjugated as primary bioassays to be excreted into the gut to facilitate the lipid absorption as well as other signaling function. In the intestines, primary bioassays will be synthesized into secondary bioassays, including DCA and LCA. However, in rodents, CDCA will be further converted to alpha-muricolic acid and beta-muricolic acid. The enzyme responsible for this conversion is CK2C17. In addition, in mice, DCA can be converted back to CA by the enzyme CK2A12. Therefore, the bioassay component in humans and mice are overlapping but not exactly the same. In addition, research from my lab showed that in addition to the classical and alternative pathway, there may be a minor pathway responsible for bioassay synthesis in mice as well. This could be minor because one may knock out CK7A1 and CK27A1, but the double knockout mice still have about 10 to 15% of bioassays in the plasma, liver, gallbladder, intestine, indicating there could be a minor pathway responsible for bioassay synthesis in mice at least. FXR, the final solid X receptor, is a major nuclear receptor responsible for maintaining bioassay homeostasis. It's highly expressed in the liver, intestine, kidneys, and adrenals. However, FXR has also been detected to be expressed in several other organs, such as endothelial cells, the eye, adipocytes. The function of FXR in those organs are not clear yet. Bioassays have been shown to be the endogenous agents of FXR. Ketodioxycholine acid turns out to be the most potent FXR activator, followed by DCA, and to some extent, LCA. We know during the last decade, amyloidic acid and UDCA has turned out to be FXR antagonists. The most potent FXR target gene in the intestine is fibroblast growth factor 1519. And this is a hormone-like fibroblast growth factor. It is with the family of FGF21 and FGF23. FGF15 and FGF19 are highly expressed in the last part of small intestine ileum. In the liver, FGF15 and FGF19 can activate FGFR4 beta-cholesterol phosphate to regulate a variety of hepatic functions. We mentioned early on, this is the most strongly-induced targeting of FXR in the gut. There is also a species difference between the human and the mice regarding FGF1519. So we know FGF15 is mainly expressed just in the mouse small intestine, but FGF19 is also highly expressed in the gallbladder and some colon cell lines. In addition, during cholestasis, human FGF19 will be highly induced in the liver, whereas FGF15 in mouse livers is not induced. Studies from our lab, as well as a few other labs in the world, has determined this tissue-specific regulation of FXR in suppressing bile acid synthesis. Specifically, they determined that FGF15 induced in the gut by FXR activation plays a major role to suppress CYP7A1 to synthesize bile acid in the liver. And then FXR activation in the liver by inducing SHIP and a few other mediators can also strongly suppress CYP8B1, but to a lesser extent, to suppress CYP7A1. Physiologically, this indicates that gut FXR signaling is important in regulating overall bile acid levels, whereas both the intestine and hepatic FXR are important to regulate hydrophobicity of bile acid. And we showed that FXR is important in NASH development, the FXR knockout mice spontaneously have a certain degree of steatosis and inflammation. In addition, after we bred FXR knockout mice with either LDL receptor knockout mice or APOE knockout mice, there's a marked increase in hepatic steatosis, ballooning, inflammation, fibrosis, indicating FXR is a protective factor in NASH development. In addition, FXR is known to be critical for liver and intestine tumor suppression. Both Dr. Moore's lab and Dr. Gonzalez's lab have shown that FXR knockout mice spontaneously develop liver cancer, and we showed that FXR knockout mice has increased susceptibility to colon cancer development. In addition, in both human and mouse HCC and colon cancer samples, FXR expression as well as function are downregulated, suggesting that FXR will be tumor suppressor. The mechanism by which FXR suppresses tumorigenesis could be twofold. We think FXR suppresses tumorigenesis first is by direct induction of the tumor suppressing genes, such as SHPE, SOX-S3, and E-cadhenin to suppress cell proliferation. On the other hand, we know FXR suppresses bile acid levels where the bile acids induce inflammation, and then subsequent induction of cell proliferation could be suppressed by FXR. Is FXR important in humans? Yes, it is. FXR is commonly downregulated in patients with liver and intestine disease. In addition, FXR polymorphism has been detected in humans. So this is a summary, nicely summarized by my student, Justin Schumacher, for the role of FXR plays in regulating hepatocytes, starling cells, aqueous sites, and immune cells. Basically, the overall function of FXR is to regulate our acid homeostasis, reduce lipid levels, reduce inflammation fibrosis, improve energy homeostasis, and decrease inflammation. Therefore, FXR is very important to regulate hepatic functions, hepatic diseases, and inflammation. And this is also a very nice summary made by Justin for the proposed roles of FGF-15 and FGF-19 in regulating bile acid synthesis, lipid synthesis, as well as immune function. So FGF-15 and FGF-19 we know is important in suppressing bile acid synthesis. It's also interesting that this mediator of this factor is very important to mediate the energy expenditure, both in peripheral tissues, as well as in the central nervous system. In addition, FGF-19 has been shown to increase muscle weight, increase muscle protein synthesis. So it seems like this factor could be important to improve energy homeostasis and treat metabolic syndrome. As we mentioned, because of the beneficial effects, FGF-15 and FGF-19 have shown to play in regulating liver functions and metabolic syndrome, including NASH. It's not surprising it's not surprising that FXR, as well as FGF-19, has been targeted as drug targets to treat cholestasis and NASH. However, all right, so this is just a brief summary of some of the leading compounds developed to target FXR or FGF-19 to treat NASH. We can say that some compounds are good to reduce liver fibrosis. However, they could increase overall LDL and the causing pruritus. Some compounds can increase insulin sensitivity. Some compounds are more potent in decreasing liver inflammation or liver fat. In addition, FGF-19 modified protein has been developed to treat NASH. Interestingly, not only FXR activators, ligands, but FXR antagonists has also been developed in the treatment of NASH. With that, we know that this pathway is important for liver physiology and the disease and has been used for drug targets. However, we have to really evaluate the short-term as long-term adverse effects associated with either FXR activation or inactivation as drug targets. In addition, we really need to determine what is the long-term effects of FXR activation or FGF-19 treatment in both animal models, as well as in humans, so that we could really balance the risk-to-benefit ratio in using this pathway as drug target. Finally, I'd really like to acknowledge all my talented students and postdocs and scientists for the study. I really appreciated the mouse models that my collaborator and I have generated to produce all these effects. Welcome to any questions you may have by the end of this symposium. Thank you. The second talk will be on intestinal regulation of hepatic cholesterol metabolism. It will be given by Tiangan Li, who is an associate professor of physiology and member of the Harold M. Diabetes Center at the University of Oklahoma Health Sciences Center. Hello, everyone. First, I would like to thank AASLD, Liver Cell Biology, SIG, for the invitation. Also, I would like to thank the chair for the introduction. As you know, cholesterol in the cells is a key component of the cell membrane structure. In the circulation, cholesterol is carried by lipoproteins. However, higher cholesterol level in the circulation is known to cause dyslipidemia, which increases the risk of cardiovascular disease. In recent years, studies have also shown that cholesterol accumulates in Nash livers, and in turn, cholesterol causes organelle stress inflammation to drive Nash disease progression. In the liver, a significant amount of cholesterol is converted to bile acids. Cholesterol-7-alpha-hydroxylase, CYP21, is the limiting enzyme that regulates bile acid synthesis in hepatocytes. Bile acid circulates between the liver and the small intestine a few times a day in the process called the enterohepatic circulation. In humans, we lose about 5% of the bile acid through fecal excretion. We also synthesize about the same amount in the liver so that the bile acid pool is maintained. Being an amphipathic molecule, bile acid has many critical physiological functions. First, bile acid synthesis is a major mechanism for cholesterol elimination. In the bile, bile acid helps solubilize cholesterol and prevent gallstone formation. In the small intestine, bile acid emulsifies dietary fat and cholesterol and lipid-soluble vitamins and facilitate their absorption. Finally, bile acid are also signaling molecules that activates nuclear receptors and G-protein coupled receptors and regulate cellular metabolism and the inflammation. Despite the important functions of bile acids, higher bile acid exposure in pathological conditions causes liver injury and also intestine epithelial cell injury. Bile acid synthesis is tightly regulated by a feedback mechanism that's mediated by the bile acid activated nuclear receptor FXR. In the liver, if bile acid level increases, bile acid can activate FXR to initiate a cascade that cause inhibition of C21 transcription and decrease bile acid synthesis. If bile acid level increases in the small intestine, bile acids activate FXR to induce an endocrine hormone called FGF-15 in mice and FGF-19 in humans. FGF-15 and FGF-19 can then bind to the cell surface receptor on hepatocytes and inhibit C21 and bile acid synthesis through a signaling mechanism. In this study, we also want to touch upon a transcriptional factor called TFAB. TFAB is a regulated through a cytosolic to nuclear shuttling mechanism. TFAB is phosphorylated by nutrient signaling such as mTOR, ERK, and AKT, and phosphorylated TFAB is sequestered in a cytosolic compartment. Upon lysosome stress or starvation, TFAB can be dephosphorylated and TFAB enters the nucleus to induce a network of genes that are involved in lysosome biogenesis, autophagy, and mitochondrial biogenesis. In the next few slides, we want to show some evidence that TFAB also regulates cholesterol and bile acid metabolism. As I mentioned earlier, intracellular cholesterol accumulation causes organelle stress and hepatocyte injury. By using a RFP, GFP, LC3 probe to monitor autophagy and lysosome activity, we can show that treating cells with cholesterol causes significant lysosome dysfunction, and at the same time, also increases the number of lysosomes in the cells. Sometime later, we further found that cholesterol accumulation also promotes TFAB nuclear translocation, indicating that cholesterol-induced lysosome stress caused TFAB activation to induce lysosome biogenesis. To further investigate the role of TFAB in regulating cholesterol and bile acid metabolism in the liver, we overexpressed the TFAB using adenovirus in mouse livers, and we found that TFAB can significantly induce CYP21 and increase bile acid pool size. This resulted in decreased intrahepatic cholesterol accumulation and also attenuated hypercholesterolemia, indicating that TFAB can induce bile acid synthesis to lower cholesterol levels. Because higher bile acid can induce FGF signaling to feedback and inhibit CYP21 and bile acid synthesis, we further studied the effect of FGF19 on hepatic TFAB function. Here we show that if we treat cells with FGF19, the nuclear TFAB is decreased, while the cytosolic TFAB is increased, indicating that FGF19 can inhibit TFAB nuclear translocation and activation. Further study shows that this is through FGF-mediated activation of mTOR and ERK signaling that cause TFAB phosphorylation and cytosolic retention. Consistent with the initial findings, when we inject mice with recombinant FGF19 to activate liver mTOR and ERK signaling, we also found that FGF19 can decrease nuclear TFAB in mouse livers and increase the cytosolic TFAB proteins. This correlates with significantly repressed CYP21 expression in mouse livers. Based on this data, we propose a mechanism where cholesterol accumulation causes lysosome stress that activates TFAB. TFAB, on the one hand, promotes lysosome biogenesis, and on the other hand, induces CYP21 to promote cholesterol conversion to bile acids. When bile acid level increases, bile acid induces FGF19 to feedback inhibit TFAB function in the liver through TFAB phosphorylation. In a small intestine, the bile acid reuptake is mediated by the bile acid transporter ASBT. It is well known that if you inhibit bile acid reuptake in a small intestine, such as by inhibiting ASBT, then the bile acid feedback mechanism on CYP21 is attenuated. This will cause induced hepatic bile acid synthesis and lowers cholesterol levels. In our model, when we treat mice with a gut-restricted ASBT inhibitor, GSK672, you can see that the fecal bile acid level is increased and the ileal FGF15 production is decreased. This also correlates with significantly increased hepatic nuclear TFAB, indicating TFAB activation. Studies from us and others have shown that blocking intestine bile acid uptake is very effective in reducing liver fat accumulation and also liver cholesterol accumulation in mouse models of methyl. These findings provide some molecular basis to further test the potential benefit of using ASBT inhibitor to treat NASH patients. However, recently, the clinical trials show that a 24-week treatment of ASBT inhibitor provided very little beneficial effect on reducing hepatic steatosis or liver injury, despite increased bile acid synthesis and decreased cholesterol. Engineered FGF19 analog is another bile acid-based therapeutic strategy for NASH treatment. Recently, studies have shown that FGF19 analog can significantly reduce liver fat accumulation and also produce a trend towards improved NASH and reduce the fibrosis in human NASH patients. However, because of the unmet need for developing a more effective NASH treatment, so we tested the idea of combining the ASBT inhibitor and FGF15-19 activation as a potential NASH treatment. To test this idea in a preclinical model, we used the mice that have been fed a high-fat cholesterol and fructose diet to induce NASH and liver fibrosis. After initial 20 weeks of feeding this NASH-inducing diet, we treated mice with either ASBT inhibitor alone, GSK, or AAV-mediated FGF15 overexpression. In the last group, mice received a combo treatment of the two therapies. Then after 32 weeks, the liver NASH and fibrosis were analyzed. The general findings of our study is that the combo therapy is very effective in reducing obesity by about 20% to 25% after 12 weeks treatment, while none of the monotherapy has an effect on weight gain. Liver pathology analysis shows that a combo therapy can reduce liver steatosis, inflammation, and cell death, and thus improve NASH activity score and also reduce the fibrosis. The effect is much stronger than either of the monotherapy. When we look at the treatment effect on bile acid metabolism, we found out that there is a remarkable reduction of total bile acid pool size by 90% caused by the combo therapy, while the monotherapy can only reduce the total bile acid pool by 40% to 50%. Further studies indicate this is because if mice are treated with ASBT inhibitor alone, the liver bile acid synthesis is induced to compensate for increased fecal bile acid loss. If mice are treated with FGF15 alone, then the reduced bile acid synthesis over time also cause reduced fecal bile acid loss. This prevents further reduction of bile acid pool. In the combo therapy, both of these compensatory mechanisms preserving bile acid pool is blocked. This allows for the reduction of bile acid pool in these mice. This reduced bile acid pool is linked to reduce the dietary fat absorption as indicated by significantly increased fecal fat loss. When we look at the cholesterol levels, we can see that the mice treated with the combo therapy showed a significantly reduced liver cholesterol and biliary cholesterol, indicating the biliary secretion of cholesterol in the small intestine is markedly reduced. Even under this condition, we still show that there is an increased fecal cholesterol loss in mice treated with the combo therapy, indicating a decreased gut cholesterol absorption that contributes to cholesterol lowering effect of the combo therapy. Finally, we showed that the serum ALT, the liver injury marker, shows a very strong correlation with liver cholesterol levels and also with bile acid pool in these mice. In conclusion, we showed that combining the ASBT inhibitor and FGF1519 signaling activation can significantly improve the treatment efficacy against NASH than either of the monotherapy. These effects, including the lipid malabsorption and higher circulating FGF levels, mimics the effect of bariatric surgery, which has been shown to be effective in promoting weight loss and reducing liver fat and improving insulin sensitivity. With that, I would like to thank many people who contributed to this work, especially two graduate students, Yifeng Wang and David Mayte, who have done a majority of the work on these projects, and also my collaborators at other universities, and also the funding from NIH. With that, I'd like to thank you for your attention. The third talk will be on gut-liver crosstalk in alcohol-associated liver disease, and it will be given by Cristina Llorente, who is an assistant professor in the Department of Gastroenterology in the School of Medicine at the University of California at San Diego. Hello. Thank you for connecting with us today. I would like to acknowledge and thank ASLT and the organizers for inviting me to talk today. Alcohol-associated liver disease is the major cause of death worldwide, and ethanol-associated liver disease is associated with intestinal dysbiosis and bacterial overgrowth as early as three weeks of alcohol exposure in mice, and it is particularly pronounced in the proximal small intestine. An increase of microbial microorganisms in the jejunum was already reported in alcoholics a while ago, and also an increase in the number of mucosal-associated bacteria in the duodenum of humans was reported. Chronic alcohol drinking disrupts intestinal homeostasis, increasing intestinal translocation of bacteria and microbial products to the liver. In the liver, it promotes chronic inflammation, exacerbating alcohol-related liver diseases. In fact, there is a therapy using a hyper-immune body cholesterol therapy containing anti-LDS antibodies that aim to reduce the permeability, and alcohol-related liver disease can be transmitted via theta microbiota transplantation from patients with severe alcoholic hepatitis to mice. Today, I will discuss major components that regulate intestinal homeostasis and how intestinal immunomicrobial integrations are doing alcohol-associated liver disease, as well as discuss some alternative therapies to attenuate alcohol-associated liver disease. The physical barriers of the intestine protect against luminal contents, pathogens, and the commensal microbiota from penetrating the body. The components of the intestinal mucosal barrier are the following, the gut microbiota, the mucous barrier, the intestinal epithelium containing different cell types, and to maintain the integrity of the intestinal mucosal barrier, we need proper tight junctions to avoid permeability. Finally, we need a proper working mucosal immune system. All of these components are altered during the alcohol-associated liver disease, suggesting a role in the pathogenic process and the onset of the disease. As an evidence of the interaction between these components, in data science, we create cytokines, such as APRIL and BAP, that can stimulate B-cells and differentiated plasma cells. They also secrete TSMP that can impact intrinsic cells. In addition to cytokines, data science can secrete antimicrobial peptides, such as defensins, bacterelectins, and catechins. Microorganisms can stimulate antimicrobial peptide secretion, and also the immune system can induce the secretion of antimicrobial peptides. For example, IL-23s can secrete IL-22 to stimulate the secretion of bacterelectins. Another example is catechins that can trap LPS and neutralize it, and for example, could be used as a therapy, alternative therapy, in the future. Defensins also promote inflammatory immune innate and adaptive immune responses, and they function as an effective chemotractors. And what happens during alcohol exposure? It's been proven that in mice and in humans, bacterelectins are tan-regulated in mice and in humans. Other components of the intestinal epithelium that can produce antimicrobial peptides are PANET cells that are located at the bottom of the crypt. Cytosine antimicrobial peptides include lysozyme C, defensins, phospholipases, cryptidins, and lectins. Interestingly, chronic binge ethanol feeding in mice has been tied to a reduction of PANET cells along with IL-17A in mice, promoting an innate inflammatory immune response. On the other hand, antimicrobial peptides such as alpha defensins have been reported to be reduced in male mice fed for eight weeks ethanol-containing laboratory diet. I just mentioned that IL-23s can secrete IL-22 to stimulate the secretion of bacterelectins. In the laminar propion, IL-22 is impaired in mice with ethanol-induced liver disease, thus compromising the epithelial barrier and decreasing the production of bacterelectins. In addition, enterocypes facilitate the immunoglobulin A transport across the epithelial barrier through transcytosis using the polymeric Ig receptor. IgA is essential to prevent microbial invasion, and the level of IgA in the large intestine and in the small intestine in mice recycling ethanol for 12 weeks was decreased. Interestingly, the levels of IgA in small intestine homogenates were increased in ethanol. Thus, they concluded that IgA plasma cells were synthesizing IgA, but not all of the IgA was being secreted into the lumen. Then, they detected the levels of PIGR in the large intestine and in the small intestine, and they found that it was actually decreased in ethanol. Another component are M-cells. M-cells can perform endocytosis and transcytosis, and present antigens to laminar-appropriate dendritic cells and macrophages to induce immunoresponses. In addition, M-cells have a partner, lymphocyte B, in the basilar lateral pocket that can help with the maturation of the M-cell. What happened during alcohol-associated liver disease? It's been reported structural changes in a model of ethanol feeding for 45 days. They detected increased mitochondrial swelling and bacular size, as well as other structural changes, but overall, it is understudied. It's been also reported a reduction in the number of payer patches in a model of 19 weeks of ethanol feeding, as well as fewer T-cells and B-cells. Endoendocrine cells secrete peptide hormones, which are very important in the gastrointestinal process. They also secrete cytokines, and they also spread receptors. G-protein receptors 41 and 43 can recognize short-change fatty acids that are very important in the inflammatory processes in mice. Chronic alcoholics present a slight reduction, a slight increase in the levels of GIP and glucagon. And in rats, it's been reported that ethanol treatment causes an impairment of intestinal somatostatin levels production. Decreased somatostatin, it is related to hypersensitivity to LBS. TOOP cells have the unusual capability of producing cytokines, such as IL-25, e-cosinase, and acetylcholine. And after an infection, it's been reported that TOOP cells can proliferate and also coagulate cells. How does this work? TUF cells produce IL-25 that induce the expression of IL-13 by IL-C2, which recruit TH2 cells that will promote the proliferation of TUF cells and coagulate cells. During alcohol-associated liver disease, more studies are needed. There is this report in colonic organoids. This is also a bioinformatic study that reported a decrease in the number of TUF cells during ethanol exposure to these organoids. This study has several limitations. It's a bioinformatic study. It is in vitro using determined ethanol concentration and is compared to a database. So it should be contrasted in the future and other studies are in need. Intestinal stem cells located at the bottom of the cliffs undergo differentiation into either the secretory or the absorptive lineages. It was reported already a long time ago that ethanol exposure reduced the proliferation in the small intestine of rats. And more recently, also in the small intestine, it was reported dysregulation and a downregulation of intestinal stem cell markers, such as VMI-1 and LGR-5 in mice. And this was mediated by dysregulation of the beta-catenin pathway. Importantly, goblet cells also participate in the secretion of antimicrobial peptides, such as resistant-like molecule and trifecta. Importantly, resistant-like molecule help with invasion against pathogens by inducing the secretion of IL-22 and recruitment of CD4-positive cells. For example, in this study, resistant-like molecule deficient mice had decreased in their CD4-positive cells as well as IL-22. Goblet cells create an important component that is mucin, and MUC2 is the major secreted mucin throughout the intestine. In this study, 47 chronic alcohol abusers and 21 controls were examined to address changes in goblet cells. And they found that during duodenal biopsies that there is an increase in the number of goblet cells, and they also contain hyperplasia. Also, later studies have shown also in the duodenum that alcoholics have a thicker mucous layer. In collaboration with Peter Starkol and Luca Macchiani from UC Levine, we have now demonstrated that alcohol disorders patients have increased number of goblet cells in the duodenum. Another important function of goblet cells is the creation of goblet cells-associated antigen passages that are abbreviated as GAPs and regulate the immune response. Usually, in the small intestine where the bacteria is lower, these GAPs are open, and in the colon, these GAPs are closed to avoid excessive influx of bacteria. These GAPs, how do they regulate the immune response? They present antigens to laminar probiotic cells. Usually, these antigenic cells control tolerogenic responses. Whether GAPs are involved in alcohol-associated liver disease is unknown and remains to be explored. We studied how chronic alcohol consumption affects GAPs. Wild-type mice were fed with liver decoagulant for 10 weeks, and we found an increase in the number of goblet cells indicated by an augmented area of MAP2 staining. We observed a reduced antigen that codified for muscarinic acetylcholine receptor 4 is the gene that promotes the opening and formation of GAPs. In addition, we found a reduction of the number of GAPs after ethanol. Based on these findings, we developed a new therapeutic approach using a muscarinic acetylcholine receptor positive allosteric modulator. It's this compound to stimulate intestinal GAP formation. Wild-type mice fed an ethanol-containing diet for 10 weeks were treated with this compound for the last 29 days. We found that the treatment with this compound abrogated ethanol-induced GAP closure and also protected mice from ethanol-induced liver injury, steatosis, and inflammation. Now, briefly, we will discuss intestinal immune microbiome interactions during alcohol-associated liver disease. Imprint mice have severe deficiencies in the mucosal immune system. The lack of gut microbiome, it is related with reduction in the number of intraepithelial lymphocytes with reduced numbers of levels of IgA, reduced levels of circulatory cells, and antimicrobial peptides, compromising the detection of pathogens. Imprint mice are more susceptible to liver fibrosis in a toxic model using thioacetamide and also CCL4. And in a model of acute ethanol exposure that mimics pinch-drinking, these mice, they had increased injury in the liver, inflammation, and induced steatosis. Now, let's discuss possible alternatives to attenuating alcohol-associated liver disease. We previously mentioned that ethanol-induced liver disease is associated with an impairment of IL-C3 and consequent secretion of IL-22. There's a few in bacterial translocation. Administration of IL-22-producing bacteria reduced liver damage, inflammation, steatosis, and bacterial translocation to the liver, and also increased the levels of rectal yeast as compared to mice fed the exogenic control bacteria. Also, there is some data in alcoholic hepatitis patients that correlates the density of IL-22 to the overall survival of patients. In another study, the treatment with a drug that is a recombinant fusion of human IL-22 also improved the survival ratio of patients. The use of antibiotics should be also considered. However, the history of their success remained controversial, as, for example, in this study, the use of paramecin in cirrhotic patients had no effect on the endotoxin concentration or liver function test. This could be due to the fact that antibiotics have an effect in the mucosal immune system, as well as some bacteria develop a system to antibiotics, and they can even open gaps in the colon favoring the translocation of bacteria. Other studies, for example, in this one, the use of ciprofloxacin as a prophylactase reduced the probability to, or increased the probability to remain free of infection and also improve the survival in these patients. And what about fecal macrobiotic transplantation? One of the latest clinical trials showed that patients with cirrhosis and alcohol use disorder, when treated with fecal macrobiotic transplantation, had lower levels of IL-6 and FBA-expanding protein. They have improved diversity and increased levels of butyric acid and isobutyric acid. Fecal microbiota transplantation, it's also related with a restoration of the intestinal immune system in the gut. Interestingly, in microbiota-depleted mice, there is a reduction in the levels of CD3-positive T-lymphocytes, B220-positive B-lymphocytes, FOXB3 T-regulatory cells, and macrophages and monocytes in the small intestine and in the colon. After fecal microbiota transplantation, we already observed a restoration of this immune cell in the colon after seven days. However, it took 28 days in the small intestine to see this restoration. Probiotic therapy has also been proven to be beneficial during alcohol-associated liver disease, but what are the highlights for the immune system? For example, lactobacillus rhamnosus GG supernatant have been shown to increase the levels of FOXB3-positive T-regulatory cells and control the levels of IL-17-positive-producing CD4 cells during alcohol binge mode. Taking ways of this talk, we summarize that the physical balance of intestine are very important to maintain intestinal immune systems disrupted during alcohol abuse and that the microbiota prevent from alcohol-induced liver injury. We also summarize avenues to prevent the disease, such as regulation of immune microbiota interactions, for example, with the use of antibiotics, fecal microbiota transplantation, and probiotics. In the future, more avenues studying immune microbiota interaction should be taken that could be beneficial to mitigate this disease. Thank you very much all for your attention. The fourth presentation will be on the liver-gut signaling in alcoholic liver disease, and it will be given by Xiaodong Ge, who's a research assistant professor in the Department of Pathology in the College of Medicine at the University of Illinois at Chicago. Good afternoon. My name is Xiaodong Ge. Today I will talk about the liver-gut signaling in alcoholic liver disease. I have nothing to disclose. The zoner of the gut liver axis in ARD has been widely studied. Indeed, a search in public retrieved 193 populations. However, the zoner of the liver-gut axis in ARD has not been studied, and there is no population. Therefore, we ask the question of whether a hepatic sterile damage associated monoclonal pattern such as high mobility group Box1 could play a zoner to connect with the gut. What do we know about the HNGB1? Upon their stimulation and the injury, HNGB1 translocates from nucleus to the cytoplasm. Its activity is secreted with disulzone as it occurs in inflammatory cells. Its pacifin is released due to apoptosis and leucocytes as it occurs in hepatocytes. HNGB1 sinkers were reached to RAS4 and others. Importantly, HNGB1 undergoes oxidized and forms a disulfide bond between 1623 and 45. Preliminary results supporting this project indicate that HNGB1 increase in alcoholic hepatitis patients, and it corrects with disease progression as shown by immunostaining in AH patients from stage 1 to 4. Moreover, HNGB1 is secreted into the serum in patients with AH. Oxidized HNGB1 is produced by hepatocyte and increase in ARD. Now we show that injection of oxidized HNGB1 works ARD as demonstrated by increasing inflammation and stertosis in mice fed with a neoproteic acid diet, overexpression of a mutant of HNGB1 that cannot undergo oxidation in hepatocytes. Prevent ARD as shown by decreased stertosis and inflammation. These results suggest that oxidized HNGB1 is involved in ARD. Interleukin-1 beta is produced by macrophage cells and play a key role in ARD. Injection of oxidized HNGB1 increase interleukin-1 beta in ARD. Overexpression of a mutant of oxidized HNGB1 in hepatocyte decrease interleukin-1 beta in macrophage cells. These results suggest that oxidized HNGB1 regulates interleukin-1 beta. Additional preliminary results indicating that ablation of the HNGB1 receptor reach in macrophage cells of parents' liver and intestine injury in ARD. As shown by HNGB1 study, there is less stertosis and inflammation in the liver and less inflammation and erosin in the interleukin-1. Furthermore, ablation of RAGE in macrophage cells decrease hepatocyte interleukin-1 beta in ARD. All in all, these data demonstrated a key donor for RAGE-syncing in macrophage cells in ARD. All preliminary results along with published work suggest that HNGB1 binds interleukin-1 beta. So we formulate the following hypothesis. In adenocortical liver disease, hepatocyte-derived oxidized HNGB1 increases and binds interleukin-1 beta. These complex syncers will reach in macrophages and aggregates of pro-inflammatory program that damages the liver. In addition, the complex causes intestinal barrier dysfunction. To demonstrate this hypothesis, the first question we asked was, does oxidized HNGB1 bind interleukin-1 beta? Firstly, we tested that binding in an in vitro reconstituted system where we incubate interleukin-1 beta with reduced or oxidized HNGB1. Immunoprecipitation for HNGB1 and immunoblock for interleukin-1 beta or the liver proved that the only oxidized HNGB1 binding interleukin-1 beta. The binding was prevented by treating with antibodies against each protein in the complex, suggested binding specificity. Next, we perform surface plasma resource. We immobilize the interleukin-1 beta on the sensor's surface and reduce on oxidized HNGB1 where ended at an increasing concentration. We demonstrated that the binding of oxidized HNGB1 is much higher than the reduced HNGB1. Then we evaluated whether oxidized HNGB1 binds interleukin-1 beta in livers for uncured adecaholic hepatitis patients. IP for HNGB1 and interleukin-1 beta and IP for interleukin-1 beta showed minimal binding of reduced HNGB1, but the binding was much higher for the oxidized HNGB1. Likewise, the complex was observed in the liver from mice. The second question is, does the complex binding reach and does it occur in microphages? To address this question, we performed SPR again. Here, interleukin-1 beta was bound to the sensor surface. A complex of oxidized HNGB1 plus ZH was ended at an increasing concentration. We observed the binding of a complex to interleukin-1 beta with a good statin of ferritin. To prove the complex binding ZH in pre-minor Kuffer cells, we performed a proximity ligation assay. In this assay, cells will grow in red if the complex binds to the receptor. In Kuffer cells from white-type mice, the complex was found. However, in Kuffer cells not in red, there was minimal red signal. To analyze if the complex is found in the liver of mice with ARD, with IP HNGB1 and IP for interleukin-1 beta and red, mice with ARD showed binding of the three proteins, suggesting that the complex binds rich in vivo. The third question is that, does the complex single-ville reach in macrophages to active or pro-inflammatory programs? Macrophages cells were incorporated with or without the complex, and RNA sequence was carried out. Besides the global signature, treatment with the complex activated the NF-CAPA-B syncope pathway and pro-inflammatory signature allowed to enhance liver injury. To validate the pro-inflammatory signature, we include macrophages cells with interleukin-beta, oxide HNGB1, or with the complex. While interleukin-beta, oxide HNGB1, increased the total TFR for under CCR4 alone, but the induction was the strongest by the complex, and it was reduced by the neutralized antibody to reach, but not to interleukin-1 beta. Similar finding was observed for the CCR4 and the CCR5 as shown on the tables. We then perform the RNA sequencing in the liver from white type and the regional cod mice. We found the same AF kappa B syndrome pathway and the pro-infirmatory signature that found in macrophagous cell treated with complex was present in mice too. This signature also validated by UNISA as shown on the table. Since the liver can also sync to gut, the first question was, is a complex found in the intestine? To address this, we performed a PLA experiment again in the intestine. We found that the complex was present in the intestine. However, it was missing in the mice that lacked HNGB1. As a second approach, we overexpressed a mutant oxidized HNGB1 in hepatocyte. The IPIB experiment demonstrated that when hepatocyte doesn't produce oxidized HNGB1, the complex is not found in the intestine. This result suggests that the sterile hepatocyte derived damp can synchro to the intestine to increase injury in ARD. The last question is, does the complex cause intestinal barrier dysfunction? To dissect this, we injected white-type and regional-card milot-cells mice with the complex. White mice developed significant liver injury as shown by stertosis and inflammation. The seronearity and liver TG and pathology score demonstrated it. Moreover, they had sterile inflammation and ulcer. However, regional-card mice proved to be significantly protected from the effect of alcohol. Since the disruption of the titan junction leads to increased gut permeability, we evaluated the expression of titan junction protein. The clotting level was significantly decreased in the triple-local mice treated with the complex, but not in the regional-card mice. As a consequence, there was enhanced gut permeability in the triple-local mice treated with the complex, but not in the other mice. Overall, this data suggests that the complex causes intestinal barrier dysfunction. In conclusion, I hope I convinced you that in ARD, hepatocyte-derived oxidized HNGP1 bisinterone beta, this complex synchro will reach the macrophage cell and the actives of poor inflammatory program detrimental to the liver. In addition, the complex causes intestinal barrier dysfunction. Future direction for this work, how does the oxidized HNGP1 increase interleukin beta? How does the complex reach the intestine? How does it regulate titan junction? Fourth, other effects of complex in the intestine also result from the activity of the poor inflammatory program. I would like to thank my PI, Professor Natalia Niorto, and the other members in our group and the source of the funding. Thank you. The fifth talk will be on hepatic adaptation to changes in the bilateral environment, and it will be given by Paul Mongaff, who is at the University of Pittsburgh, Maygall Center, where he's the endowed chair for experimental pathology. He's also a professor of pathology and medicine at the University of Pittsburgh and the founding director of the Pittsburgh Liver Research Center and NIDDK-funded DDRCC. Hi, good afternoon. I'd like to begin by thanking the organizers for giving this opportunity to talk about some of our work. I'm going to talk today on hepatic adaptation to changes in the bile acid environment. As we know, gut-liver access is very important in liver homeostasis, and bile acids are a major component and effector of this communication. Bile acids are produced by hepatocytes. They make their way through the biliary canals into the bile ducts and then head into the small intestine, where they are absorbed in the terminal ileum and eventually make its way through portal circulation back to the hepatocyte. In the intestine, the bile acids are known to perform detergent function that is relevant in lipid emulsification and absorption. The entire hepatic circulation is quite an efficient process, and there's minimal loss of bile acids via feces every day, such that only a little amount of bile acids have to be replenished by de novo synthesis from cholesterol in the liver. Also, bile acids are filtered by the glomeruli and can be reabsorbed in renal tubules. We also know now that bile acids can signal in the liver and gut through these receptors like FXR, PXR, vitamin D receptor, and TGR5 to regulate many different biological processes, including metabolism, liver regeneration, and inflammation. As you can imagine, bile flow via both the intrahepatic and the extrahepatic ducts are really an essential part of the interhepatic circulation. And what is quite interesting is that the bile has to be initially generated from the hepatocytes, has to go into the biliary canals through these small bile ducts, then it makes its way into the intermediate and then larger bile ducts, eventually going into the extrahepatic biliary tree before it goes into the duodenum. What is also interesting is that the developmental origin of the intrahepatic bile ducts and the extrahepatic bile duct is, in fact, distinct. What I show here is that the common progenitor cell, which is in the ventral endoderm, gives rise to two different types of stem cells, one hepatoblast and the other one pancreatobiliary progenitor. And while the intrahepatic bile biliary tree originates from hepatoblast, the extrahepatic biliary tree's origin is from the pancreatobiliary progenitor. So what we have been interested in the lab is to really understand the signaling pathways that regulate the decision-making of the hepatoblast, especially in generating intrahepatic biliary tree. For that, we study the role of yes-associated protein 1 in liver development. As we all know, YAP1 is part of the hipposignaling pathway, and what I show here are two states of the signaling pathway, hippo-on on the left and hippo-off on the right. When hipposignaling is turned on, YAP is actually phosphorylated by these kinases and in their phosphorylated form are either undergoing degradation or are bound to proteins like 14333 and hence sequestered in the cytoplasm. Whereas if the hipposignaling is off, YAP signaling is active, it translocates into the nucleus where it binds to the TAD group of transcription factors to turn on different target genes that are known to play a role in cell proliferation, stemness, actin cytoskeleton regulation, and others. The question that we have asked is what is the role of this protein YAP1, especially in hepatoblasts, which are precursors, as I said, to both hepatocytes and intrahepatic biliary tree. For this, we utilized fluxed YAP mice and bred them to FOXA3 CRE. FOXA3 CRE, unlike albumin CRE, is active early in the liver development, as early as embryonic age 9.5, and FOXA3 is expressed at high levels in these hepatoblasts. And that was really the decision, that was the reason why we chose FOXA3 CRE to breed to our YAP1 fluxed mice. When we did this, we were successfully able to delete YAP1 in hepatocytes as well as in cholangiocytes, as can be seen in the right panel, where the YAP immunohistochemistry is shown in a wild-type animal on the left. These mice with absent YAP in epithelial compartment of the liver are born at normal Mendelian ratio, but as you can see here in the picture, the knockouts are significantly smaller than the wild-type, and they continue to lag behind in growth throughout their adulthood. Also, these are obviously yellow and hence appear to be jaundiced. When we look at their serum biochemistries, indeed, we find there is a significant increase in hepatocyte injury marker like ALT, but intriguingly, this was observed only at early postnatal stages, and this ALT was sort of normalized at 13 weeks and 8 months. Interestingly, cholangiocyte injury as seen by alkaline phosphatase and also by hyperbilirubinemia is evident throughout in the knockouts all the way up to 8 months, and at that stage, there is a significant increase in mortality. So, these animals, they're alive, they're born at normal Mendelian ratio, but they are unable to survive beyond 8 months. When we looked at their histology, what was obvious to us was that the knockouts had significant presence of this biliary in fox, which is typically seen when bile acids are not able to be dumped through the bile and the extrahepatic and intrahepatic biliary system. Also, there was increased periportal fibrosis, and there was also increased inflammation throughout, but especially in the periportal region. When we look more carefully for a ductile marker like CK19 or SOX9, while in the controls, we saw very nice luminal ductular structures in the periportal area, there was absolute absence of that ductular structure in the knockouts of FIAP1. You can see here the SOX9 positive cells that are just randomly present, not forming any tubular structure, and hence this complete absence of well-formed bile ducts or intrahepatic bile ducts. To confirm the absence of bile ducts, we decided to use tissue clearing and ribbon confocal microscopy, and while I won't have time to kind of go over the exact process, I just wanted to show that we take the whole lobe, we clear it, and then stain it for cytokeratin 19 in red. As you can see here, we see in the wild-type animal very nice gallbladder and this extra intrahepatic biliary tree with all its branches. The resolution of this technique is fantastic and really can show in great detail the hierarchy of your intrahepatic biliary tree. On the other hand, in the knockout of FIAP1, we do not see the presence of intrahepatic bile ducts at all, and the only thing that we see here is the presence of a gallbladder, and when we are looking for CK19 positive intrahepatic biliary tree, there is absolute absence of it. This was also confirmed by traditional India ink injection through the extra hepatic biliary tree, which can really nicely decorate the intrahepatic bile ducts in the control animal, but absolute absence again in the FIAP1 knockout. Finally, we cannulated the extra hepatic biliary tree to monitor the bile flow, and while we see bile flow in the wild-type, there is absolutely no bile or very little bile that was present in the knockouts, showing a complete absence of, again, the intrahepatic biliary tree. So the question that we asked then was, how are these mice alive? They have zero intrahepatic biliary tree, and they're able to be alive for the next eight months after birth. So for this, we performed bulk RNA-seq and did transcription factor enrichment analysis on the differentially expressed genes, and what we identify is several signaling pathways that are affected, and one of the top hits is, not so surprisingly, alterations in the bile acid metabolism, and factors like LXR, RXR, and PPAR-alpha were the transcription factors that were most downregulated or altered in the FIAP1 knockout mice. How do these knockouts then adapt to this, and they do so by three different mechanisms. One is by decreasing or affecting the bile acid biosynthesis. As you can see here, there are several genes that are responsible for bile acid synthesis, and several of them, including CYP7A1, CYP27A, etc., are significantly downregulated. Please note that on the y-axis, this is a log scale showing a notable downregulation in various BA biosynthesis genes. As a result of this, we think that the bile acids in the knockout are still kept under check. They were still elevated in the liver of the knockout as compared to the control. However, if these biosynthetic pathways were not affected, we would see significantly higher levels of bile acids in the liver. Interestingly, we continue to see a significant increase in total bile acids that made out into the serum. So, we started looking at some of the genes that are responsible for encoding bile acid transporters. There are three broad categories of transporters, including the ones that are sinusoidal transporters or influx transporters that are bringing in bile acids from the sinusoidal blood into the hepatocytes. When we examined their expression, we found significant decrease in the expression of several key transporters, including NTCP, OATP1, OATP4, and OCT1. We also looked at the expression of the apical transporters, which are moving bile acids from the hepatocytes into the apical or the canaliculi. We find, again, a significant decrease in the expression of ABCG5 and ABCG8. Finally, we looked at the bile acid transporters that are dumping bile from the hepatocytes back into the sinusoid or the efflux transporters. When we examined the expression, we found significant upregulation of MRP1 and MRP4, whereas MRP3 did not show a significant difference. To confirm that this was indeed happening, we used intravital microscopy to show that while in the control, the bile canaliculi show very nice green staining, which is actually indicating the bile flow and also how nicely the bile is sequestered from the sinusoidal blood, which is in the red here. In the knockouts, you see a significant mixing of bile and blood, suggesting that the bile is being dumped into the blood. Finally, the last part of the adaptive process was alteration in the species of bile acids. There were several bile acids that were altered both in the liver as well as in the blood of the knockouts as compared to the wild types. I won't go into the details, but if you calculated the hydrophobicity index of all of these, we found a significantly lower hydrophobicity of the bile acids in the knockout, suggesting yet another protective mechanism. The more hydrophobic a bile acid is, the more toxic it is to the liver. As a result of all of this, including that the bile is less toxic, it's being dumped back into the blood, and its synthesis is reduced, the liver is able to adapt and the animal is able to survive. But what happens after eight months of dumping this bile back into the blood is that the kidneys of this animal begin to fail, and you'll see significant cystic and obstructive chronic disease, as can be seen in the right lower panel. So this is my summary. We find that YAP1 loss from hepatoblast results in absent intrahepatic bile ducts reminiscent of allergy syndrome. There is absence of hepatocyte tubular transdifferentiation, which is one of the repair mechanisms, but it's YAP-dependent, and hence in the absence of YAP, we don't see it. But what we do see is that the livers adapt dramatically, but through metabolic reprogramming. And just my final slide to thank the individuals and funding, and the credit for most of the work that I showed today goes to Laura Molina, who's an MD-PhD student who just finished her PhD and has gone back to medical school. So thank you again for your attention. Thank you for joining the Liver Cell Biology SIG. We hope you like the program, and we will see you next year.

Liver Cell Biology

Gut-Liver Interaction

Liver Disease

FXR-mediated Communication

Bile Acid Synthesis

FGF15 and FGF19

Bile Acid Homeostasis

Metabolic Syndromes

Cholesterol Metabolism

Alcohol-associated Liver Disease

YAP1 and Bile Duct Formation