Hi, good afternoon everyone, and welcome to the basic science debrief. I'm Heather Francis and I'm from the Indiana University School of Medicine, and I'm going to share with you some of the highlights from the meeting that I picked up from all the outstanding work that's been presented over the last few days. So I have no disclosures, of course. So the first abstract I want to cover is glycolysis and hepatic stellate cells induces fibrogenic EV release and promotes liver fibrosis, and this was also highlighted as a foundation abstract award winner. Authors have previously demonstrated that activated HSCs increase the release of fibrogenic extracellular vesicles or EVs, nanosized particles, and that these impart cell to cell interactions. Activated HSCs also display high glycolytic profile and hexokinases, or HK, that catalyze the first committed step in glucose metabolism. So here the authors have used an HSC selective knockdown of HK2 and induced fibrosis by using carbon tetrachloride, and also isolated primary HSC derived extracellular vesicles. And what the authors have found here is that fibrogenic signals increase glycolytic HK2 levels, and HK2 induces the transcription of RAB27A, a protein that's involved in EV release, and HK2 also promotes liver fibrosis in vivo. And the key takeaways from this abstract were to demonstrate a novel role for HK2 and glycolysis in liver fibrosis, and a new mechanism involved in HSC derived EV release. And this proposal might help us increase our understanding of the pathobiology of liver fibrosis. So in our second abstract, this one is titled, Disregulated Sphingolipid Metabolism is Associated with Brain Dysfunction in the MDR2 Knockout Mouse Model. So now we're talking a little bit about something a little bit different about the brain and the liver axis and how this might be regulated. So these authors have the hypothesis that cholestatic liver injury is induced by dysregulation of sphingolipid metabolism, and this can contribute to neurological disorders. So these authors use both age and gender match wild type and MDR mice to examine the impact of cholestatic liver injury on brain function, and also sphingolipid metabolism. And the main findings of the study here were that the severity of the cholestatic liver injury of MDR2 knockout mice was associated with infiltration of CD11B positive myeloid cells in both the brain and in the liver. And the mRNA levels of key inflammatory mediators like IL-1 beta and MCP1 were also significantly increased in the liver and within the brain. And the key genes involved in sphingolipid metabolism were markedly upregulated, especially SPHK1, SMPD2, and ASAH2 in the brain and ASAH1 and SGPP1, SMPD3 within the liver. So S1PR2 expression was also found to be significantly upregulated in both the brain and the liver. And the conclusion that was made from this study was that cholestatic liver injury induced loss of sphingomyelin and the imbalance of sphingosin-1-phosphate and ceramide may contribute to the brain dysfunction, particularly memory loss in this model of PFC injury. The next abstract that I highlighted is the deletion of TNFSF10 trail in myeloid cells augments ductile reaction, fibrosis, and inflammation in a DDC diet-induced model of cholestatic liver injury. So in this study, the authors have demonstrated that chronic liver injury is associated with ductile reaction. They assess tissue sections for ductile reaction by immunostaining for the markers CK19 and MIC1. And the first panel shows the CK19 for the control mice, and the second panel shows the staining for CK19 in mice that were fed the DDC diet. And then the third panel indicates the DDC diet, MIC1 staining. So the comparison of the wild type and the trail mice shows that there is a greater expression of both CK19 and MIC1 in the trail-deleted mice as compared to wild type. The violin plots presented here depict the number of CK19-positive cells observed per field of view and provide an overall view of the ductile reaction across the entire liver lobe. And the median number of CK19-positive cells was 30 for both genotypes, 70 for the wild type on DDC, and then 92 for the trail-deleted mice on the DDC diet. The more than 40% of the field type greater than 100 cells per view, and these observations were also confirmed by quantification and immunostaining. So this data confirmed that in this diet-induced cholestatic model, there is greater expansion of ductile reaction in the trail-deleted mice. So when we look at their graphical summary working model, we can see here in the wild type mice that we have trail that then binds to the trail receptor, and this is normal ductile reaction that's found within the wild type mouse. However, when there's a myeloid-specific knockout of trail, there's an upregulation of ductile reaction, and the bile ducts begin to express more trail receptor, enhancing this ductile reaction and inflammatory process. So in my next one, I highlighted endotoxin-stimulated hepatic stellate cells augment acetaminophen-induced acute liver failure and non-STAR condition directly and by influencing KUFER cells. So most studies that challenge, that study APOP or acetaminophen-induced toxicity challenge rats or mice following starvation. However, injury will occur in humans even without starvation. So therefore, the authors investigated the role of HSCs and APOP-induced liver injury in mice and rats that were under a FED condition. And so by H&E, we can see here that there is decreased inflammation in the stellate cell-depleted model when it was challenged with either LPS, APOP, or LPS with APOP. In addition, the authors found that TNF-alpha, IFN-beta, and IFN-gamma were all increased in the rats that were challenged with LPS or APS or LPS and APOP together. However, in the stellate cell-depleted model, these factors were also reduced. So the key takeaways here are that HSCs can increase inflammatory response of KUFER cells to LPS and that mitigation of APOP-induced liver damage in HSC-depleted mice suggests that HSCs play a critical role in APOP metabolism by hepatocytes. And my next abstract is Interferon-gamma induces cholestatic liver injury in the R-del-PBC model by increasing hepatic MHC-2 expression and altering bile acid metabolism. So in this abstract, the authors aim to determine if INF-gamma plays a direct cellular role in the pathogenesis of cholestatic liver injury by studying the R-del mouse model, which is a mouse model of PBC. And the authors found that MHC-2 expression was increased in both hepatocytes and cholangiocytes in the R-del female mice. And hepatic mRNA and protein expression of genes involved in bile acid homeostasis were also decreased in the R-del, FXR, BSEP, NTCP, and CYP7A1, all shown here by real-time PCR. And IFN-gamma treatment of human hepatocytes and or normal human cholangiocytes increased recruitment molecule expression like CCL2, CXCL10, ICAM-1, while decreasing these other factors, FXR, NTCP, CYP7A1, CYPAB1 expression. So interferon gamma induces cholestatic liver injury in the R-del PBC model by increasing this. And the co-culture of R-del CD4 positive T cells with hepatocytes significantly increased IFN-gamma induced CXCL10 secretion versus wild-type CD4 positive T cells. So here are the key takeaways. Our IFN-gamma plays a key role in autoimmune cholestatic liver injury by increasing MHC-2 expression, sensitizing hepatocytes and cholangiocytes to immune cells, and directly downregulating hepatocyte genes involved in bile acid homeostasis. So the pathogenic role of IFN-gamma in human PBC and the therapeutic potential of targeting the signaling pathway warrants further investigation. All right, next we'll move on to KUFR cells and hepatic immunosuppression. This one was titled, Irregulatory Loop Consisting of AKT, YY1, MIR206, and beta-catenin LEF1 in KUFR cells Drives CCL22 Production in Hepatic Immunosuppression. So here the authors have presented some fantastic data showing there's an activation of AKT in KUFR cells of patients with hepatocellular carcinoma. And there's actually an overexpression within these HCC tumors when these were compared to normal tumors. Also, MIR206 drives M2 to M1 shift within KUFR cells, suggesting inflammatory cascade. And the authors also found that when they did KUFR cell-specific expression of MIR206, they were able to prevent HCC development, shown here with their gross tumor pictures and also by H&E histology. However, when they depleted CD8 positive T cells, this actually impaired the ability of MIR206 to prevent HCC and where they had recurrence of the disease. The key points in this study are that activation of AKT in RAS drives M2 polarization of KUFR cells. AKT RAS impairs biogenesis of MIR206 in KUFR cells via YY1. AKT drives CCL22 overproduction by phosphorylating beta-catenin in M2 KUFR cells. And then there's a regulatory loop consisting of AKT, YY1, MIR206, and beta-catenin that maintains CCL22 overproduction, which then recruits Tregs via the CCR4. So KUFR cell-specific expression of MIR206 fully prevents HCC, while 100% of AKT RAS mice died from HCC within six to eight weeks post-injection. So depletion of the CD8 T cells partially offset the inhibitory effect of MIR206 HCC. And the overall conclusion of this study is examination of MIR206 within KUFR cells may play an important role in hepatocellular carcinoma. The next one is selected targeting of alpha-4-beta-7-MADCAM1 suppresses hepatic fibrosis progression by decreasing the recruitment of immune cells to the injured liver. So in this study, chronic inflammation was examined, which can be orchestrated by injury-associated hepatic immune cell recruitment and is the primary driver of fibrosis and advanced chronic liver diseases. The authors evaluated the role of the signaling pathway and regulating T cell recruitment to the injured liver and its role in fibrogenesis. So mice were treated with carbon tetrachloride to induce liver fibrosis, and then either treated with an anti-alpha-4-beta-7 or an anti-MADCAM1 antibody or controls. Also, they also looked at liver biopsies from patients with ALD and NASH. And you can see here that when they treat with carbon tetrachloride, there's an induction of hepatic fibrosis, shown also here by alpha-SMA, that is reduced when these mice were treated with the alpha-4-beta-7 antibody. And similarly, we also see here that the hepatic fibrosis and H&E demonstrates a more normal profile when the mice were treated with the MADCAM1, or this is a knockout, MADCAM1 knockout flux animal. So the key takeaways from this study are that higher infiltration of alpha-4-beta-7 T cells in the livers of NASH and ASH-associated cirrhotic patients was found, that alpha-4-beta-7 MADCAM1 access is involved in the recruitment of T cells to the fibrotic liver. And activated HSCs are the primary cells that express MADCAM1 in the injured liver. And activated HSCs promote liver injury by recruiting T cells to the liver. So blockade of alpha-4-beta-7 or MADCAM1 may represent a novel therapy for preventing progression of liver fibrosis in chronic liver disease patients. Now we'll move into an alcohol study. This one is knockout of biliary neurokinin-1 receptor, or NK1R, ameliorates liver phenotypes and alcohol-associated liver disease, or ALD. And in this study, the authors have demonstrated that human ALD patients have increased levels of substance P within their serum when this is compared to the controls. Furthermore, there's also an increase in ductile reaction and an increase in NK1 expression that's found primarily within cholangiocytes in patients with ALD when these were compared to the normal patients. And in addition, in an animal model, alcohol-induced ductile reaction, shown here, liver injury, hepatic steatosis, inflammation, and fibrosis were all decreased when NK1 receptor knockout mice were fed ethanol when they were compared back to their wild-type controls. So you can see here by HNE, oil red O-staining, CK19, CD68, and serious red that with the loss of NK1, which is found primarily within cholangiocytes, ameliorates ALD phenotypes in mice. And so the authors conclude here that inhibition of biliary NK1R signaling access may be an additional important approach for the management of the ALD phenotypes. Here's another alcohol study. Reduction in alcohol preference and intake is transmitted through colonization of germ-free mice with stools from AUD patients that received fecal microbiota transplants. So in this study, the objective was to determine if drinking behavior improvement in humans after FMT could be transmitted to mice and to also determine the microbial taxa associated with these changes. So the authors here used stools from humans pre- and post-FMT were used to colonize male germ-free mice and drinking behavior over two days were studied and linked with the microbiota composition. This is all demonstrated here in this graphic here. And the main findings from the study was that there was a reduction in total and binge alcohol intake in mice that were colonized with post-FMT stools versus FMT stools that was linked with ruminococca and higher microbial diversity. So the authors conclude here that FMT may be a promising therapy for reducing alcohol intake and craving transfer of microbiota rich in this bacteria and are associated with lower intake and preference for alcohol in the germ-free mice. The next one I'm going to talk about is the VEGF-A induces cholangiocyte-driven liver regeneration in mouse models of acute and chronic liver injury, and I want to highlight that this was also a Foundation Abstract Award winner. So in this study, the authors aim to harness the regenerative potential of cholangiocytes by promoting their conversion to hepatocytes to treat liver disease. And the hypothesis here is that VEGF-A, a ligand for VEGFR2KDR, delivered with nucleoside-modified mRNA complex to lipid nanoparticles promotes cholangiocyte to hepatocyte conversion and also restores liver function. So they use a cholangiocyte-specific knockout model using this Cre19 tomato mice that were used to fake trace cholangiocytes. Using AAVs, this TBGP21 was administered to mimic impaired hepatocyte proliferation that is found in many human liver diseases. And following CDE to induce injury, after two weeks of consecutive injections of the nucleoside-modified mRNA encoding to VEGF or controls, these were administered through intraorbital sinus to efficiently transfect the hepatocytes. So the main findings here were that transient VEGF-A expression and secretion by hepatocytes via liver-targeted mRNA LNP delivery robustly and significantly increased the number of cholangiocyte-derived TD tomato-positive hepatocytes, as well as significantly reverting steatosis and fibrosis and the chronically injured livers. And so we can see here, we have a NASH model here, and by steatosis, I believe this is ODIPI staining. You can see here the poly-C controls and the lipid spots. And then after VEGF treatment, the fibrosis as well is reduced. And also, the number of tomato-positive hepatocytes was significantly increased that were derived from cholangiocytes following these treatments. So this study reveals a novel therapeutic benefit of the safe and non-integrated VEGF-A mRNA LNPs to harness cholangiocyte-driven liver regeneration to treat chronic and acute liver diseases in which the hepatocyte-driven liver regeneration is compromised. So importantly, this project introduces non-integrated mRNA LNP currently used for COVID-19 vaccines as a clinically safe tool for VEGF delivery to potentially treat human chronic liver diseases. Our next one is gut microbial factors activate intrahepatic B cells to promote non-alcoholic steatohepatitis via innate and adaptive signaling. And this was also selected as a Foundation Abstract Award winner. So in this study, the authors wanted to investigate the mechanisms of intrahepatic B cell activation during NASH. And so they used microbial antigens, looked at MYD88 signaling and B cell receptor signaling, and how these might contribute to the inflammatory liver via production of the pro-inflammatory cytokines. They also examined the instigation of intrahepatic T cell-mediated inflammation and the promotion of liver fibrosis. So here we have our hepatocytes and inflamed hepatocytes as we drive the liver from a healthy liver through NAFLD and into NASH. Of course, we have the presentation of B cells that increases as we progress from healthy liver into NAFLD. And this is also going to alter the intestinal epithelium. And here we have the lumen and the healthy microbiota, and how this can also be changed, of course, as we see this activation of the MYD88 and the toll-like receptors that can, of course, induce leaky gut syndrome and inducing changes in the microbiota as we progress from NAFLD into NASH. And then the consequences here are the fibrosis. We have increased inflammation IL-6 TNF-alpha, and, of course, we have the INF-N gamma and the CD8 T cell interactions. So the takeaway points from this study were that NASH-associated changes in gut microbiota composition leads to the accumulation and activation of intrahepatic B cells. And intrahepatic B cells promote liver inflammation in a process involving the innate adapter molecule MYD88, and intrahepatic B cells also promote liver fibrosis, but the mechanisms of this are still unknown. So what are some avenues of future research? The author wanted to highlight NASH-associated gut microbiota, interaction with B cell activation, and, of course, the eventual liver fibrosis that's found in NASH patients. And the author also wanted to add that this is a study that was just recently published in Hepatology, so congratulations to all of these authors on this significant findings. And that's all I have for the basic science debrief. I hope to see everybody in person next year in Washington for AASLD 2022. Please be safe and take care of yourselves, and don't forget to watch everything on demand. Thank you.

liver fibrosis

brain dysfunction

cholestatic liver injury

alcohol-associated liver disease

gut microbiota

liver regeneration