Thank you, Dr. Singhal and McLean for inviting me. These are my disclosures. Before I start talking about the drugs and the treatment we can do for alcoholic liver disease focusing on gut-liver axis, I just want to briefly summarize what we currently believe, how the gut microbiome and the microbes contribute to alcoholic liver disease. So you have, I don't know if you can see my pointer, apparently not. Do we have a pointer here? It doesn't work. Now I got it. Okay, sorry. So you start off with a healthy gut microbiome. And under conditions, we start to understand you end up with a dysbiotic alcohol-associated dysbiosis. And this is characterized, first of all, by qualitative changes. So you get compositional changes in the gut microbiota. But then you also get overgrowth. And we now know that this is not only restricted to bacteria, but also to fungi. And this dysbiosis then has different effects. One of the effects, obviously, they produce many of these microbial products, which we commonly know as LPS. Or we have shown that also beta-glucan from Canada contributes to this. And the dysbiosis also contributes to changes, for example, in other metabolites, such as bile acids. So we get increased secondary bile acids. On the other hand, the microbiome metabolizes some of these metabolites in the intestine. And they produce other metabolites that could be, for example, deleterious, such as acetaldehyde. But they also have a decrease in beneficial metabolites, such as butyrate. So one of the key issues and key points that we know contributes to alcoholic liver disease is that you get this disruption of tight junctions. So the paracellular space between the intestinal epithelial cells opens up and now allows many of these microbial products to translocate through this space to the portal way and reach the liver, where they can contribute to liver inflammation and also to hepatocyte death. In addition, dysbiosis triggers this subclinical inflammation in the lamina propria, which contributes to these tight junction disruptions. Acetaldehyde from the metabolism, from ethanol, contributes to it. And also a decrease in butyrate all contributes to this, what we commonly call, gut leakiness. In addition, we know that secondary changes in the secondary bile acids lead to changes in FXR signaling and also changes in the biofeedback towards the liver and FGF19. And I have a separate slide at the end of my talk showing how this contributes to it. So now, how can we approach alcoholic liver disease from a gut perspective, from a microbiome-centered perspective, to start treatment? We can obviously target the dysbiosis. Dysbiosis we can target very simply with antibiotics, probiotics, or fecal microbiota transplantation. We can use, on the other hand, we can use anti-LPS, so just to prevent the LPS from translocating. And this has been done in a clinical trial using boa and colostrum with anti-LPS in severe alcoholic hepatitis patients on steroids. This enrollment, as far as I know, has been closed, so we are awaiting the results from this treatment. We know that zinc and IL-22 are very important, especially IL-22, not only in the liver, but also it contributes to mucosal homeostasis. So IL-22 can be protective in the intestine and can prevent potentially antimicrobial peptide production, can prevent bacterial translocation. And finally, we have another target of therapy is FXR here. We can use FXR agonists and also FGF19. So in the following slides, instead of just walking you through this, you know, the list of all these drugs, what I have thought is, you know, I give you a different prospect of how we can treat the gut microbiome and show you now precise approaches to restore host microbiome homeostasis, how we can achieve this with the overall aim, obviously, that we target alcoholic liver disease. So one of the first concepts is we can use Baxis drugs. So essentially what we are doing is we can analyze healthy and diseased patients. We can analyze the gut microbiome. We can see what differences we have. And then we can use these defined mixtures of microorganisms to reprogram the microbial ecology to restore intestine homeostasis and essentially to improve alcoholic liver disease. The most crude and primitive way is obviously doing this with fecal microbiota transplantation, where you just wipe out the entire microbiome and replace it with a donor stool. This has been done, as you can see here, in a very small trial with NS8 in steroid ineligible patients with alcoholic hepatitis. You can see that daily FMT for eight days via nasal jejunal tube improved survival as compared to a historical control group. And this is currently confirmed and assessed in three independent prospective trials. Dr. Bashar also used fecal microbiota transplantation in 10 patients with decompensated cirrhosis and hepatic encephalopathy. He pre-treated these with five days of antibiotics followed by an FMT enema and then compared this group to a standard of care group. And you can see it's not moving forward. And you can see here that in the FMT group, you get an improvement in the hepatic encephalopathy score, but not an improvement in the standard of care group. I'm sure many of you have been aware of this brief report, which was recently published in the New England Journal of Medicine, where drug-resistant E. coli have been transmitted via fecal microbiota transplantation. One of these patients, in fact, died. So I think in the future, we obviously need to optimize the screening method for our donors and include such drug-resistant bacteria. A next option, which we can also use, is probiotics. So we just give beneficial bacteria. This has been done in a multitude of trials. I have just summarized here some of the more controlled trials of double-blind RCTs. You can see that mostly they have been conducted in thyroidic patients with alcohol abuse. This first trial also included alcoholic hepatitis patients. The problem with these trials is, first of all, they have all a very mixed outcome. And it's not clear what the real endpoint in most of these studies are. And the second problem is that any of these trials uses a different combination of probiotics. So I think in the future, we need to try to reproduce the results from another published trial to make sure that this is reproducible. Currently, there is a larger trial ongoing with alcoholic hepatitis patients and treatment with LGG. So lactobacillus ruminosus, GG versus placebo. Another, and now we are going more to precise precision medicine approaches now, we can use engineered microbes to restore microbial ecology. And what we can do is we can engineer these bugs to either produce beneficial metabolites, or what has been done, we can engineer bacteria to reduce waste products. And in our case, in this case, it was used to reduce intestinal ammonia. There was actually very good preclinical evidence that this approach would work. And then this press release from Synlogic, which made the bacteria, came out recently. And they showed that this randomized, double-blind, placebo-controlled trial with 23 patients with cirrhosis and elevated blood ammonia, no evidence of blood ammonia lowering or changes in any of the endpoints versus the placebo was achieved. So we are awaiting the results in the publication from this study to see why this very promising novel probiotic has failed. We have used a different approach, and this is in the preclinical stage. At this point, we have bioengineered lactobacillus ruteri to produce IL-22. IL-22 is one of these very beneficial, as I mentioned before, very beneficial cytokines in the intestine, which restores gut homeostasis, which stabilizes the gut barrier. So we have used this in a preclinical mouse model of alcohol-induced liver injury. We have used the PBS group. We have used the isogenic L-ruteri, which is just one we used to overexpress IL-22. And these are the IL-22-overproducing bacteria. You can see that we can increase the antimicrobial molecule production in this mouse model. We can decrease bacterial translocation to the liver. And most importantly, at least to some degree, we get an improvement in alcohol-associated liver disease. Dr. Shah will tell you later on about his trial with systemic IL-22, but I'm very sure that this might not only have an effect on the liver, but it might also have a very beneficial effect on the gut barrier by stabilizing this gut barrier function. Finally, we can use what we can not only do. We cannot only add something like FMT probiotics or engineered bacteria. Instead, what we can also do now, we can very specifically eliminate deleterious microbes. And there are different approaches. Obviously, we can use antibiotics, which are not very selective. We can use bacteriocins, which are small molecules produced by bacteria that affect and kill other bacteria. And we can use bacteriophages with the aim overall that you restore intestinal homeostasis. In this case, that you eliminate this deleterious bacteria. There is currently one trial ongoing in France, in Lille. I think the enrollment has been closed, so we are also very soon to hear the results from this trial. They used Augmentin with standard treatment in severe alcoholic hepatitis patients. One trial has been published as an abstract at ESL last year. This was Rifaximin in alcoholic hepatitis patients. This was a multi-center pilot trial with 24 patients with severe alcoholic hepatitis. 1200 mg per day of Rifaximin for 90 days of standard treatment with steroids. Primary endpoint was presence of bacterial infections at 90 days in mortality. And this was compared to a historical control group. You can see that in the Rifaximin group, there were patients who had less infections. They had a lower survival and also the infection-free survival was higher. However, none of these endpoints reached statistical significance at this point. That might have been also an issue of underpowering. Coming now to very precise medicine approach. What we have recently shown is that when we compare the gut microbiome in patients with alcoholic hepatitis and compare it to controls in alcohol use disorder patients, you can see that you get a very prominent increase, a 3000-fold increase in Enterococcus faecalis. However, this did not correlate with any of the disease outcomes or with any of the mortality. Instead of looking who is there and who is not there, what we did, we focused on the functionality of this bacteria. We said, what are they doing? We did a very careful analysis and what we found is they produce one toxin. This toxin is called cytolysin. As you can see here, controls for alcohol use disorder patients are colonized, or a very small percentage of these patients and subjects are colonized with cytolysin-positive Enterococcus faecalis. While in the case of alcoholic hepatitis patients, you see that 30% of these patients are colonized with cytolysin-positive strains. This correlated very nicely with disease outcome and mortality. You can see that most of the cytolysin-negative patients actually survived, while most of the cytolysin-positive patients died within 180 days. Hazard ratio for this is 14. What we thought, how can we precisely now eliminate this cytolysin-positive bacteria in the intestines? We obviously had to switch to an animal model. The approach we took is we used germ-free mice, which we colonized with either stool from a cytolysin-positive alcoholic hepatitis patient or from a patient who was cytolysin-negative, also with alcoholic hepatitis. What we thought about is how we can eliminate this was bacteriophages. What are bacteriophages? You can see one scheme here. They have a head and a tail. These bacteriophages, with their tail, dock onto very specifically certain strains of bacteria. They can inject the DNA. They can replicate inside. Then they lyse this bacteria and essentially destroy this bacteria. You have a very selective elimination of certain bacteria. We used and we grew and isolated phages that are targeting cytolysin-positive enterococcus faecalis. When we did this, you can see here two patients that we have colonized. If you use this phage approach, you can decrease alcohol-induced liver injury in this humanized mouse model. You can decrease steatosis. But most importantly, what you can do is you can also eliminate or at least decrease the toxin that goes to the liver in this humanized mouse model. It's a very precise and precision medicine approach to focus on deleterious bacteria. This is my last slide. Focusing again, coming back to this bile acid, FXR, and FGF19 signaling. Bile acids are produced in the liver, as you know. They are going via the bile duct into the small intestine, where they help digesting your food and help the absorption of lipids. In the terminal ileum, they are being absorbed. They can enter the enterocytes, can activate FXR signaling, and turn on transcription. In humans, you get FGF19 release, which reaches the portal vein. Then the liver shuts down the bile acid synthesis. This is its negative feedback mechanism. FGF19 has not only effects on the bile acid synthesis, but it also has very beneficial effects in general on metabolism and lipid metabolism. It has regenerative capacity, and it also, unfortunately, also has some tumorigenic activity. FXR, we know, is also beneficial for glucose and lipid homeostasis, immune responses, and insulin signaling. So, one clinical trial with obete colic acid with patients with moderate severe alcoholic hepatitis has been, unfortunately, halted by the FDA when there was this issue with the toxicity of obete colic acid in cirrhotic patients. So, this study has not been completed yet. So, this is my summary slide. I believe that intestinal dysbiosis represents a very attractive target for therapy. We have very promising results from pilot studies. We are awaiting, obviously, results from ongoing RCTs. Targets for therapy need to be precisely evaluated, and to give you a little bit of an idea about this, I think we need to consider various factors. If we look at the gut-liver axis in the microbiome, we need to classify, really, microbiota-driven subtypes of disease. It might not be that every alcoholic liver disease patient is, in fact, driven by gut barrier dysfunction. For example, we need to validate these new targets in the host microbiota interaction with the aim of restoring host microbiome homeostasis. And then we can use, as I have shown you, bugs as drugs, phages as drugs, and examples I have not given you, but there are examples out there in the literature, drugs from bugs, and we can also drug the bug to inhibit metabolic activity in this bacteria. Thank you for your attention.

gut microbiome

alcoholic liver disease

gut-liver axis

dysbiosis

precision medicine