Thank you for attending the basic science debrief. My name is Hermit Mulhey, and I am a professor of medicine and an associate professor of physiology at the Mayo Clinic in Rochester, Minnesota. I'm also the chair of the basic research committee of the AASLD. I have no disclosures relevant to the content of this presentation. Just a high-level overview of the basic science programming at the liver meeting digital experience. We had a Hans Popper basic science state-of-the-art lecture, a Hyman J. Zimmerman hepatotoxicity state-of-the-art lecture, the basic science symposium, the basic research workshop, the clinical, which was very translational this year, research workshop. We had basic science SIG programming, a total of 631 abstracts in the basic or translational track, 24 poster categories, and 8 parallel sessions. There are also 12 meet-the-experts sessions classified as basic or translational. Continuing with the basic science symposium, this was held on 11-14-2020. The title was mechanisms of alcohol-associated liver disease, molecular, cellular, and systemic crosstalk. This was organized by Drs. Nagy, Ding, and Shah. And some of the high points were a discussion of the diversity of immune cell responses in alcoholic hepatitis, elucidation of specific molecules that are gut-derived metabolites and how they mediate extracellular, how they mediate alcoholic hepatitis, and then also the potential of extracellular vesicles as a biomarker for alcoholic hepatitis. The basic science workshop was held on Sunday, November 15, 2020. The title was gut microbiota from descriptive studies to targets of therapy, and it was organized by Drs. Schnabel and Bajaj. And really, they took a deep dive into the good and the bad of gut microbiota. There was a lot of discussion about this concept of co-occurrence, where bile acids, short-chain fatty acids, and microbiota changes are interconnected, and really, a long discussion on FMT. Though the programming from these two sessions will not be available on demand, the question and answer session, which really included a discussion of these key points, will, and I encourage you to attend that. The Hans Falker basic science state-of-the-art lecture was titled Modeling Development and Cancer Using Liver and Biliary Organoids, presented by Professor Hutch, and this is available on demand. The Hyman J. Zimmerman Hepatotoxicity State-of-the-Art Lecture is titled Hepatotoxicity from Checkpoint Inhibitors, Biologicals, and Small Molecules, and it will be delivered by Professor Bjonsen on Monday, and I anticipate will be also available on demand. SIG programming is, in fact, available on demand. The hepatotoxicity SIGs programming was titled Basic Mechanisms of Hepatotoxicity and Their Clinical Implications, and the talks covered hepatocyte mitochondrial and oxidative stress response, bile acid homeostasis, adaptive immunity in the formation of neoantigens, and epigenetic and genetic pathways. The liver fibrosis SIG programming, NASH fibrosis from matrix to medicine, the talks encompassed molecular pathways, immune system, lipid metabolism, hepatocyte cell death, biomarkers, and what's next in NASH fibrosis. Liver cell biology and hepatic diseases SIG programming was titled Cellular Senescence in Liver Disease and Aging, and the talks touched upon basic concepts, aging in liver cells, and biliary senescence. These are the basic science parallel sessions. Some of them will be available on demand, others won't. We'll now dive into the posters. So diving into HCC, this study, characterization of the transcriptional landscape of human NAFLD-associated HCC using single nucleus RNA sequencing aimed to take hepatic resection specimens and then following a process of tissue lysis, tissue homogenization, filtration, isolation of nuclei, nuclear stain, leading to single nuclei RNA sequencing. And this study was important because single cell RNA sequencing has allowed for the study of complex mixtures of tissues and cells, and they tried to utilize fresh tissue, which is optimal for single cell studies, and using single nuclear study, you can also use archived frozen material from which nuclei can be isolated. This particular process has been performed in brain tissue, and in this study was done in liver tissue. And they found that cell type and gene-specific heterogeneity are observed. Shown here is uniform manifold approximation and dimension, UMAP dimension reduction to visualize the complex data in two dimensions, and the graph on the left highlights three specific patients indicated as 1, 2, and 3, and you can clearly see that there is significant heterogeneity per patient. And the graph on the left compares tumor tissue with non-tumor tissue, highlighting heterogeneity by tumor status, and really the single cell RNA sequencing studies are increasingly helping us perform a more molecular classification of cancers, and a molecular phenotyping, and perhaps informing treatment responses to cancers. The second study I picked in cancer is abstract number 601, matrisome genes from hepatitis B induced HCC are unveiled. In this study, the goal was to understand how hepatitis B induced extracellular matrix remodeling can facilitate the onset and progression of HCC. By analyzing these available gene sets, they show that differentially expressed matrisome genes discriminate tumor from non-tumor samples with HPV infection, shown here as a heat map in A, and by principal component analysis in B. Altogether, they found 63 down-regulated and 17 up-regulated matrisome genes in GSEA 55092 and 121248 microarray data sets. Figure 2 shows the key matrisome genes in HPV induced HCC, and immune pathways shown in black, cancer pathways shown in light gray, and ECM pathways are shown here in gray. How these data would compare with matrisome genes in HCCs of other etiology will have to be tested, and which of these might be a therapeutic target will also be tested in future studies. We will shift gears now and speak about fibrosis and matrix, but continue with the theme of single cell RNA sequencing. Abstract number 324 looked at single cell RNA sequencing, which reveals FHL2, which stands for four and a half limb domains protein 2, as a stiffness regulated molecule to promote liver fibrosis. In this study, fibrosis was induced by carbon tetrachloride administration, and cell subpopulations were identified by single cell RNA sequencing using the 10x genomics platform. In figure A, you can see by tSNE plots, the single cell RNA sequencing data from CCL4 in red and healthy in turquoise. Hepatic stellate cells are highlighted here as the green and purple populations, showing us the two clusters of hepatic stellate cells, and both clusters are present in healthy as well as CCL4 treated livers, though their distribution is a little different. The heat map in C shows us the signature genes in cluster 0 are different from cluster 1, and both collagen 1 alpha 1 and collagen 3 alpha 1 are highly expressed in cluster 0, which thus contains the fibrogenic hepatic stellate cells. They next go on to show that FHL2 is highly expressed in hepatic stellate cells in cluster 0 that express high levels of collagen, and by immunofluorescence, they saw that these FHL2 positive hepatic stellate cells are located in collagen-rich stiff areas of the liver. Looking at abstract number 313 by Sado et al., titled Fibrosis Resolution Involves More than Collagen, Key Role of MMP12 in Fibrosis Recovery in Mouse Liver. The aim of this study was to investigate the metabolism of the total hepatic ECM during fibrosis resolution using a degradomic analysis of the plasma. The methods were to induce fibrosis resolution after CCL4 administration for four weeks. Total plasma peptides were analyzed by 1D mass spec analysis, and quantitative and qualitative features were extracted and analyzed using Peak Studio. The main finding here was that MMP12 and elastin were the most persistent ECM protease pair during fibrosis resolution, as you can see in panel A, which shows the normal liver, the fibrotic liver, and recovery from fibrosis liver, and you can see MMP12 was significantly increased in the fibrotic liver, but persisted in an increased form in the recovery from fibrosis conditions. In B, they show the top-down analysis of the degradome and how from this data, they identified a new player in fibrosis resolution, which is shown in C, and they conclude that MMP12 and its substrates could play key roles in fibrosis resolution and analysis of the degradome, and notably plasma degradome could lead to new biomarkers to predict mechanism and outcome in HCC fibrosis. We're going to shift gears now and talk about several abstracts from alcohol-associated or induced liver disease, depending on whether they're from humans or mouse models, and maybe due to the themes of the basic science symposium and the basic research workshop, there were a significant number of excellent abstracts that dealt with alcohol models. This is a poster number 246, or abstract number 246, excuse me, by Aberia et al, also using single-cell RNA sequencing to show us that Kufra cells undergo anti-inflammatory changes in the early phase of alcohol exposure. In this study, they found that Kufra cells have major heterogeneity with 10 clusters that fall into two broad clusters, and you can see the TISNI maps here, and a black line drawn through them, dividing them into class 1 that are more anti-inflammatory and class 2 that are more pro-inflammatory in terms of gene expression. Cluster 0, which is shown here, is the largest cluster. The table on the right shows us cluster, characteristic gene expression of that cluster, and predicted function of the macrophages in that cluster in this model of alcohol-induced liver injury. Let's look at the data on the left of this slide, where they show that during the early phase of alcohol adaptation, Kufra cell numbers initially decline, but due to apoptosis, and then are rapidly restored. The photomicrographs are for F480 immunostaining. A naive liver is shown in the leftmost panel. Day 3 after alcohol administration and day 11, and the reddish-brownish dots are staining for F480. This is quantified in the bar graph below. You can see there's a significant reduction by day 3 and normalization by day 11. The heat maps on the right show us that over this period, gene expression undergoes an anti-inflammatory shift. The largest cluster, which is cluster 0, and represents 40% of the cells, shifts from a pro, which is shown here in the middle, to an anti-inflammatory pattern, and by day 11 looks more like cluster 1. We'll shift gears now and talk about pro-resolving lipid mediators in alcoholic-associated liver disease. This is abstract number 42, showing that resolvin D1 and its receptor FPR2 signaling attenuates alcohol-associated liver injury via suppression of hepatic STAT1 activity and pyroptosis. In this study, the major findings are that resolvine detreatment is protective in alcoholic liver disease and reduced STAT1 activity and pyroptosis. They show that FPR2 knockout mice develop exacerbated liver injury and inflammation in an ALD model. And the resolvine D1 FPR2 signaling reduces STAT1 activity and pyroptosis in KUFA cells, bone marrow-derived macrophages, and liver. And hepatic resolvine D1 levels are negatively associated with STAT1 genes and pyroptosis markers in AH liver tissue. So this is a cartoon of their findings in mouse models on the left and humans on the right. And in this model of alcohol and LPS, resolvine D1 can reset or alter the inflammatory milieu of the liver to lead to resolution of inflammation and less liver injury. And in human severe AH, there is a depletion of resolvine D1 signaling, which corresponds with increased STAT1, pyroptosis, and AH severity. And this is particularly cool because the resolvine pathway can be targeted for therapy. Moving on to the next abstract, number 40, chronic plus acute binge alcohol induces steatohepatitis and fibrosis in mice with hepatic deficiency of tuberous sclerosis 1. In this study, wild-type mice or TSC1 knockout mice were treated with alcohol. And you can see injury in the leftmost panel and steatosis in the graph below were significantly increased in the knockouts in comparison with the wild-type. So the red triangles in each set correspond with the alcohol-fed model. And you can see that both the ALT was significantly higher in the TSC1 knockouts without any impact on the amount of steatosis or fat in the liver. The second finding from this study was that there is a much greater ductular reaction in the knockouts. I left out the wild-type images for brevity, but you can see with CK19 immunostaining that there is significant ductular reaction in the alcohol-fed TSC1 knockout mice. And the panel on the right shows that macrophage and neutrophil infiltration is also reduced in this TSC1 knockout mouse. F480 immunostains are shown in the top panel for macrophages. Myeloperoxidase immunohistochemistry is shown in the bottom panel for neutrophils. And these are quantified in the graphs below. What's shown here, fibrosis correlated with injury and inflammation as well, confirming a novel role for TSC1 in alcohol-induced liver injury. The next alcohol abstract is abstract number 41 in which they study the hepatic FKB binding protein 5. And this to be an alcohol-induced gene, which is significantly elevated in chronic plus single binge ethanol-fed mice, and that the deletion of FKBP5 ameliorates the hepatic phenotypes of alcoholic liver disease. So mRNA expression is shown in the graph on the left, and you can see that it's significantly elevated in wild-type mice-fed alcohol, which is this light gray bar. The western blot shows a significant increase in FKBP5 protein level in the wild-type E, which is ethanol conditions with none detected in the knockout. And if we focus our attention on the photomicrographs, the wild-type E shows significant alcohol-induced steatosis, neutrophil accumulation, which is not evident in the FKBP5 knockout ethanol-treated livers in the right-most panel. This abstract caught my attention because they go on to study the mechanism by which this happens and find that alcohol-induced FKBP5 expression is mediated by methylation at its promoter region and conclude that the reduction in methylation levels of FKBP5 by alcohol at its promoter region led to an increase in the expression of FKBP5 mRNA and protein expression. We'll shift gears to talk about this abstract, number 242, which is on hepatic single-cell sequencing reveals a biliary progenitor-like transcriptional program in patients with severe hepatic hepatitis, or SAH. In this study, their aim was to examine alterations in epithelial cell function in SAH at a single-cell resolution. So they performed single-cell RNA sequencing on cells from three healthy donors and recipients five of living donor liver transplantation for SAH. And the sequencing was performed using the 10X Genomics Chromium 5' gene expression chemistry. They used published data of adult fetal hepatocytes and progenitors to derive markers of mature and immature cells. They found that there were three major clusters of cells, as you can see by the UMAP plot on the left, and progenitor cells are depicted in blue, cholangiocytes in pink, and hepatocytes in green. Epithelial cells were identified by expression of typical structural markers like KRT18 and function-specific genes, and the liver progenitor populations were characterized by high expression of LRMP, HEPA-CAM2, and PLCG2. The violin plots on the right show that the hepatocyte score in SAH, which is shown in blue, was far lower than the cholangiocyte score, leading to the conclusion that there is much more of a biliary progenitor-like program in patients with SAH. We'll now shift gears and talk about regeneration. This was abstract number 167, titled Dynamic Epigenetic Programming Enables Functional Diversification of Hepatocytes During Liver Regeneration. In this study by Chen et al., partial hepatectomy was used in mice to model liver regeneration, and they combined single-cell ATAC-seq and RNA-seq analysis with immunohistochemistry to analyze the hepatocyte compartment in the regenerating liver at single-cell resolution. Their notable findings were that a fairly large subpopulation of hepatocytes undergoes a reprogramming process that moves them into a less differentiated fetal-like state where they acquire markers of liver progenitors. And then the gene expression profiles of transitioning hepatocytes are enriched in cytokines like TNF-alpha and IL-6 signaling and also hedgehog signaling and signaling processes that these pathways regulate like inflammatory signaling and epithelial to mesenchymal transition. They found that a unique chromatin signature marks the most fetal-like cells and is characterized by increased accessibility to transcription factors, and you can see the transcription factor motifs deviation score in the image on the left. And some of these key effectors are effectors of hedgehog like GLEE1 and GLEE2, of EMT, of ductal type D differentiation, and other transcription factors associated with even more primitive liver progenitors. They show that immunostaining validates expression of these factors in the nuclei of hepatocyte tick-appearing cells in regenerating livers after partial hepatectomy as shown in the images above for YAP1, SMED2, SOX9, GLEE2, and GATA6. They conclude that the fetal-like state is transient and rapidly disappears after partial hepatectomy, maximal at 48 hours and pretty much gone by 96 hours, and that hepatocytes sort of divide and conquer in order to maintain liver-specific functions while meeting the dramatic increase in a proliferative response. The next study we're going to talk about for regeneration is one of the late-breaking abstracts LO6 titled Microfibril Associate Protein 4 or MFAP4 as a Potential Regulator of Liver Regeneration and Disease by Iakovleva et al. And in this study, they conducted RNAi screen for the identification of new therapeutic targets in hepatic regenerative medicine, and in addition, for a better understanding of the process of liver regeneration and disease. Their approach to the RNA sequencing, RNA interference, RNAi sequencing, and the model which was thioacetamide or TAA treatment is shown here. By deep sequencing, using an shRNA pool that contained 250 shRNAs against 89 genes, they were able to identify MFAP4 as a top candidate, as you can see in the graph on the right. They go on to study this in various models and identify this as a new potential therapeutic target as MFAP knockdown accelerates liver regeneration, MFAP knockdown attenuates chronic liver damage-related liver fibrosis. They found that MFAP knockdown attenuates NASH-related liver fibrosis, and in vivo knockdown of MFAP impacts mTOR and p38 signaling. They further show that MFAP is conserved in human cells. We'll shift gears now and talk about the gut-liver axis. Sorry for the slide issues. My computer seems to be not cooperating. This is abstract number 10, that branching amino acids protect cirrhotic liver damage through the suppression of LPS binding protein, TLR4, STAT3 activation, and eficalis translocation. In this study, the authors show that branching amino acid treatment recovered the intestinal dysfunction by resulting in a reduction in eficalis infiltration in the liver and suppression of liver inflammation via suppression of the LBP-TLR4-STAT3 activation and reduction of hepatocyte cell death and recovery of intestinal dysfunction. Thus, branching amino acids may have a broad beneficial effect in cirrhosis. This is abstract number 61, longitudinal metagenomic analysis of fecal microbiome in infants. This is a study of infants with biliary atresia by Yang et al. And they show that at diagnosis compared to normal babies, biliary atresia patients have more abundant opportunistic pathogens, which are proteobacteria and bacilli, and less protective bacteria, bacteroides and clostridia. At diagnosis, biliary atresia patients or babies lack of pathways that are associated with the synthesis of glutamine or glutamate. You can see the pathways that show a significant change in function. And for example, the urea cycle and the L-citrulline pathways are enriched in normal controls, which are associated with glutamine and glutamate molecules. And at three months after surgery, infants with biliary drainage had enriched pathways related to fatty acid biosynthesis. You can see here responders greater than non-responders that are fatty acid related are shown in the solid orange dots. And patients with better bile drainage had increased fatty acid metabolism three months after surgery. So again, highlighting more than just what happens to the microbiota, but a functional consequence of the changes in microbiota. Lastly, I would like to express gratitude to all of the presenters who shared their slides and allowed me to modify them. I'm indebted to AASLD staff, in particular Jessica Jessup and Stephanie Grimsby, who were very helpful in putting together this talk. I'm grateful to Gavin Arteel for sharing slides from his talk last year. And I'm very grateful to the basic science brigade that functioned as scouts and helped me identify interesting posters and parallel session talks. I will add that the breadth and depth of basic and translational science and hepatology remains impressive and I would like to remind you that many sessions are available on demand. Thank you.

basic science programming

liver meeting digital experience

alcohol-associated liver disease

single-cell RNA sequencing

gut-liver axis

therapeutic targets

molecular mechanisms