Okay, good morning everyone. It's a pleasure for me to present the introductory lecture to this workshop today. So I'm going to try to convince you that the microbiome contributes significantly to liver disease and the reasons for which we should study it and move progress forward in this area to better understand hepatology. I have no disclosures relevant to this presentation, so I'll start off by going over what the microbiome is, its role in health and disease, what has been the state of knowledge in the context of liver disease, and then finally what are the gaps that need to be filled in order to move progress in this area. So what is the microbiome? Microbiome is an ecological community of commensal symbiotic and pathogenic microorganisms within a body space. Microbial cells outnumber host cells in the human by tenfold, approximately 10 to the 11 microorganisms in our body, non-pathogenic in general, symbiotic with the host, which is true with all animals and plants, and it is a stabilizing force for maintenance of health. I'll focus specifically on the intestinal microbiome for the sake of this talk, and I think most of our workshop is going to be dedicated to really the intestinal microbiome itself. So the intestinal microbiome is very diverse, 14,000 prokaryotic species over that level known, consists of trillions of bacteria, and there are over 1,000 bacterial species, fungi, protozoans, and viruses also live symbiotically. What's important to know are the genome proportions as well as the richness of these genomes, and finally who's there and what can they do. So moving on to the role of the intestinal microbiome in health and disease. Commensal bacteria do provide significant benefits to the host in terms of health. So protective functions, pathogen displacement, nutrient competition, production of antimicrobial factors, structural functions such as barrier fortification, induction of IgA production as well as immune system development, and then finally metabolic functions, which are critical in terms of maintenance of human health. And so these metabolic functions include sucrose degradation, carbohydrate, amino acid, fatty acid metabolism, de novo synthesis of essential vitamins such as vitamin K, cobalamin, biotin, et cetera. And what's very interesting is that the bioactive compounds produced as a result of microbial degradation then serve to maintain the epithelium, the intestinal epithelium, help in immunomodulation as well as nutrition. This is a figure illustrating the axis of the host microbiome metabolic interaction. So again, as I mentioned earlier, if you look at these compounds here that are produced as a result of microbial degradation, they then travel to the liver via the portal vein and then are metabolized to various other compounds that then serve to affect systemic metabolism. And additionally, these compounds may also travel through lymphatic vessels and then affect organs systemically. And interestingly, one of those is the adipose tissue, which will affect leptin production. So this is all very important in terms of the metabolic axis in the human. So what is it that affects the intestinal microbiome? And this is very important to realize in the setting of health versus disease. So in liver disease particularly, so fatty liver disease as well as alcoholic liver disease, diet and exercise are very important. The presence of intestinal inflammation, particularly in the setting of PSC, aging, medications such as antibiotic intake, acid suppressants, metformin, these are all factors that will affect the health versus disease of the microbiome. So enteric dysbiosis refers to alteration of the microbiome composition. And what we know is maintaining the relative abundance of each component within the intestinal microbiome is crucial to maintenance of health. If enteric dysbiosis occurs, this will result in disruption of integral networks within the host and consequently result in disease. So Hippocrates famously said in 340 BC that all disease begins in the gut. Well, not quite, but certainly this is established for some diseases including chronic metabolic disease. And so here you see the healthy situation, which is where there's mechanisms involved in the crosstalk between the microbes and the host. So you have commensal bacteria that are normal, result in higher mucus layer thickness, production of antimicrobial signals, short-chain fatty acids such as butyrate and propionate, which then result ultimately in secretion of GLP-1. And then if disturbed, if all of this is disturbed, so what happens is then you have disruption of these signals and there's possibility of the intestinal barrier becoming more permeable, resulting in systemic release of LPS, lipopolysaccharide. And this ultimately will result in effects of metabolic endotoxemia, such as increased systemic inflammation, increased energy intake, increased blood glucose, insulin resistance. And so this is a phenomenon that's been now established in the setting of metabolic disease. And this is from a very nice review paper published by Patrice Canney in GUT 2018. So next I'll move on to the gut-liver axis and its role in liver diseases. So why the liver? Well, the liver is in closest contact with the intestinal tract given portal blood flow. It contains various types of cells that all will likely be affected by these microbial peptides and signals. And they also serve to carry out the functions of the liver in protection from infection, metabolism, et cetera. Many compounds come from nutrient digestion and the liver is exposed to a substantial amount of bacterial components and metabolites. And these contribute to host homeostasis. Gut-liver communications are essential to health. So the gut barrier is a border between the intestinal lumen, portal blood, and the liver. Prevents bacterial translocation and translocation of microbial-derived products. Intestinal factors regulate bile acid synthesis, glucose, and lipid metabolism. The liver as well performs essential functions to maintain intestinal health. So it transports bile salts and antimicrobials to the lumen, maintains eubiosis by controlling bacterial overgrowth, and liver metabolites can affect the gut barrier positively with butyrate and negatively with acetaldehyde. So what is the state of knowledge in terms of the microbiome and its role in liver diseases? It's been known over the years that there is an improvement in liver disease and complications of cirrhosis with prebiotics, probiotics, and antibiotics. And these have suggested a causal role for the microbiome. And I think the recent years have brought about lots of data with the various tools and techniques that we now have available, so Dr. Tilg will be elaborating on those in his lecture. Enteric dysbiosis has been seen in alcoholic liver disease, fatty liver disease, primary biliary cholangitis, primary sclerosing cholangitis, as well as hepatocellular carcinoma. What's generally seen is increased enterobacteriaceae and decreased acromantia muciniphila. Additionally, these studies have demonstrated small intestinal bacterial overgrowth, translocation of the PAMPs, which are microbial peptides, intestinal and systemic inflammation. So what the recent data is showing is that dysbiosis may influence the gradual progression of liver diseases, inflammation, fibrosis, and dysplasia over time. And mouse studies are really starting to provide mechanistic insights into how enteric dysbiosis, including specific taxa, affect liver disease progression. So I'll focus on a few particular liver diseases where there's lots of data that's been generated and even causality has been demonstrated. So this is from a beautiful review paper by Dr. Tilg, who is our next speaker. And this figure demonstrates how the microbiome contributes to NAFLD progression. So patients will ingest decreased fiber, higher consumption of saturated fat, and specific xenobiotics, which can all alter the IM composition and gut barrier function. And this will result in leaky gut. As I mentioned, when that intestinal barrier is disturbed, this will result in translocation of bacteria and these microbial-derived peptides, including LPS. And this will then serve to generate systemic inflammation and disturb hepatic gene expression and stimulate lipogenesis. And causality has been demonstrated, actually, in NAFLD by fecal microbiota transplantation into germ-free mice from both mouse and human. And these are two references down here. So certainly causality has been demonstrated in recent studies. Alcoholic liver disease. So the data here have demonstrated reversibility of dysbiosis with abstinence from alcohol. Causality has been demonstrated also by FMT into germ-free mice. So there's one study where FMT from patients with severe alcoholic hepatitis resulted in severe hepatitis in mice when the stool was transferred into germ-free mice. There's a second study where FMT from ALD-resistant to ALD-susceptible mice prevented liver injury in recipients. Then evidence for IM for the intestinal microbiome and other liver conditions, PBC and PSC. Patients have been reported to have enteric dysbiosis, but causality not yet established. Cirrhosis. So there's a very nice study, actually, from Dr. Bajaj, who is one of our speakers in our workshop. And this is an excellent study wherein FMT from cirrhotic patients into germ-free mice resulted in higher degrees of neuroinflammation and neuronal activation. With respect to hepatocellular carcinoma, limited clinical studies have detected an overgrowth of E. coli, which is part of the Enterobacteriaceae. And animal studies have demonstrated changes in tumor burden with prebiotics, probiotics, and antibiotics. So finally, I'll conclude with gaps in the understanding of the intestinal microbiome and liver disease. So I think we need to go beyond characterization. There have been a number of studies using 16S RNA sequencing, and Dr. Tilde will talk about this a bit later. But basically, this is sequencing the variable regions, particularly V4, but there are other variable regions, in order to determine the bacterial taxa and species present in a sample. And I think one of the tools that we can use, actually, on already generated data is inferred functional metagenomics. So there are tools such as Pycrust and Pyfilin that operate based on machine learning algorithms. And basically, in this particular tool, Pycrust, what they did was they used the human microbiome project data to connect and associate 16S RNA sequencing data to metagenomic sequencing data. And then based on a given 16S RNA sequencing sample, one can then deduce the actual functions that are represented within a particular microbiome. And so this is a tool that I'll provide as an example of what we've done in my lab. So we looked at 30 liver transplant recipients and looked at longitudinal functional changes over time after transplant to examine the contribution to long-term complications post-transplant. And we determined that over time, there was an increased representation of metabolic pathways and metabolic pathway-associated genes over time. As you can see here, there's a whole list, and this was based on Pycrust analysis. We then also looked at various other types of genes, like cancer susceptibility, infection susceptibility. I won't go into the details, but just to give you the idea that it is possible to use tools such as this on 16S RNA sequencing data that already exists out there to better understand the gene functions that are represented. So going beyond simple characterization. And what we then did was validate this data in animal metagenomic sequencing data that we had previously generated. So this was from a paper that we published a few years ago, and we looked at metagenomic sequencing data that we had generated in rats exposed to immunosuppression and looked at these pathway changes that occurred in the patients versus those in the animals. And we found these common pathways represented. So we were able to validate an association study using animal metagenomic sequencing data. So what about causation? So I spoke about this briefly earlier about the NAFLD and ALD studies where causation has been demonstrated with FMT into germ-free mice. And so Cox postulates state that the microbiome should cause disease. Well, Cox postulates state this for infection in general. But basically, applying this to the microbiome, the microbiome should cause disease when reintroduced into the healthy, susceptible animal model. And so here, what this figure shows is basically an obese mouse, its microbiome being transferred into germ-free mice, and then ends up becoming obese. So I think these kinds of studies have been extensively performed in metabolic disease, and I think we need to be pushing that in hepatology further. So the key take-home points from my talk, what are the gaps in study of the intestinal microbiome in hepatology? I think most work to date in hepatology has been limited to characterization and association studies. I think we need to move more towards mechanistic studies establishing causation. Liver disease is dynamic, so is the microbiome. So there's a need to perform longitudinal studies. Also looking at the functional impact, how does the microbiome alter hepatic gene expression, metabolism, newer techniques such as metatranscriptomics, metaproteomics. These are all tools that will, in future, provide further insight, I think, especially when we bring together those different layers of data to provide further insight into liver disease. Subsequent speakers are going to delve into the diagnostic biomarkers, as well as therapeutic applications in the intestinal microbiome as applied to liver disease. So I think, overall, there's lots of work to be done, and I hope that our workshop will provide you with the tools that you can then use to apply this to your own research program and help move this field forward. So thank you very much for your attention, and I hope you enjoy the rest of this.

microbiome

liver disease

intestinal microbiome

gut-liver axis

NAFLD