 I'd like to start off by thanking the organizers for allowing me to be part of this discussion. So when Lita asked me to give this talk, she wanted me to talk on the impact of the gut microbiome on host epithelial functions and responses. And I'm going to do that, but largely through inspiration received from Owen White yesterday, I decided to go rogue last night. And I shortened my talk to allow more time for discussion of the gaps, needs, and challenges. I'm still going to talk about what my charge is, but towards the end I think you'll see that I'm going to go outside of that charge to talk about things and I think broadly apply to research and hopefully will advance some of our thinking and approaches to the study of human microbiomes. So this slide just is a compilation of examples of how critical gut epithelial functions are impacted by microbes. And they include a variety of functions, for example barrier function, development, wound healing, immune functions, cytoprotection, transport, autophagy, and proliferation in apoptosis. And I think that you will appreciate that many of these processes are quite essential for allowing the gut epithelium to be the interface between us and the outside world or in the case of our own microbiota or inside world. They provide very important functions in separating us, but also there are selective barriers so that we absorb critical nutrients, water, electrolytes that we need to sustain life. Now what I'm going to do in the next few slides are show some specific examples and these are just examples that come from a literature that is replete with both mechanisms and processes that have been identified that mediate how microbes affect epithelial function. Shown on the left is just a stain for KI67 which is a marker of cellular proliferation. And you can see that in the control, the colonic crypt is shown here and in the lower half of the colonic crypt are most of the proliferating cells. But if you take this mouse and you treat this mouse with a cocktail of broad spectrum antibiotics over a period of a few weeks, you see a dramatic change. This is a mucosal atrophy with a significant decrease in cellular proliferation. Now proliferation is only one side of that coin and if you look at apoptosis, which is also important in maintenance of epithelial homeostasis, I referred you to this study by Brent Polk and his group where they identified two peptides that were secreted by lactobacillus GG, which they called P40 and P75. And both of these panels show staining for apoptosis, which is in brown. And you can see that after treatment of the mucosa with TNF, you get a significant increase of apoptosis. But if you pre-treat that tissue with either P75 or P40, you can significantly abrogate the induced apoptosis. The next line is an example, one of many, but this one comes from our laboratory of conditioned media from Bifidobrev, which protects both mouse dejunum as well as human Keiko II epithelial cells, protects their barrier function against reactive oxygen species. And that's illustrated here, that is, here are the controls. And in this case, we're measuring paracellular flux using tritiated mannitol. When you add the ROS, you can see that the barrier function breaks down, both here and here. But if you pre-treat these tissues with conditioned media from Bifidobrev, you can mitigate that effect on barrier function. Now, one of my favorite examples, and I think is a very good example of host-microbe interaction, is the induction of heat shock proteins. So heat shock proteins are highly conserved proteins. And in the gut, they're physiologically expressed in two regions of the GI tract. One is the stomach, and the other is the colon. And I don't think that that's by chance. These are the two most hostile areas of gastrointestinal tract. And as I will show you, these heat shock proteins are absolutely essential for maintenance of intestinal homeostasis. But there's something very unusual that I wanted to highlight by showing you this Swiss roll of imbunostaining for one of the heat shock proteins, HSP25. So a Swiss roll is basically where we remove the colon, and then we cut along the mesenteric border, we roll it up, and then we slice it like a Swiss roll. And what that allows us to do is look at protein expression, both along the vertical axis as well as the longitudinal axis. And I think you can appreciate by the brown staining that there is a... Most of the heat shock protein is expressed by the surface colonocytes. That is exactly the cells that are in direct opposition to the luminal fluid and gut microbes. The other thing I think you can appreciate is that there's a gradient of expression, which is largest in the proximal colon, but then begins to dissipate and is almost undetectable by the time you get to the rectum. Now, one of the things that we noticed immediately was that, or we thought of immediately, is that perhaps the reason that these proteins are being physiologically maintained is because they're getting cues from microbes. And in support of that, if you look in a germ-free mouse or if you antibiotic treat a mouse, what you find is that you get an abrogation of the expression of these heat shock proteins. Now, implied from that is that if this gradient, if it is due to cues from microbes, then there must be a gradient or there must be a heterogeneity of microbes that are found in the proximal versus the distal colon. And I think that through studies that we've done, both of 16S as well as metagenomic profiling, this indeed is true. And that's shown on this slide. So this is a study where we did a colonoscopy in a healthy human subject, and this was done in a patient or a volunteer that underwent this procedure without colonic lavage. That is, we tried best not to perturb the communities of microbes in the colon. By doing so, I think that you can appreciate that the metagenomic profiles of the mucosa-associated microbiota are quite different between the right colon and left colon. And each slice of these pie charts represents different subsystems, functional subsystems. The fact that these heat shock proteins are absolutely important for maintenance of intestinal homeostasis is illustrated here. So if you take a wild-type mice and treat them with dextran sodium sulfate, an agent that will induce colitis, wild-type mice typically will develop inflammation, but it will spontaneously resolve over about a two to three-week period. On the other hand, if you have a mouse where the gene for HSP70 is deleted, these mice do very poorly. They actually exhibit much more severe colitis. And after several treatments of DSS, they go on to a chronic-like colitis that is sustained. And the colitis is very typical of what we see in human ulcerative colitis. There is a presence of cryptapsis, branching crypts, and also inflammatory infiltrates. These mice also develop the typical IBD metaplasia to carcinoma sequence of cancers that we see in our patients. Now, a lot is known, and the literature is quite robust with regard to how microbes affect the host side. What is really less well-known are what are the mediators that microbes use to impact epithelial function? A few have been identified, and I list them here. They include things like quorum-sensing molecules, innate ligands, short-chain fatty acids, and lactic acid, hydrogen sulfide, chemotactic peptides, and certain metabolites. But this is certainly not all-inclusive list, and it's only the tip of the iceberg. And discovery and identification of other agents awaits better technology to be able to detect them. So this is a segue to my slide on what are our gaps in our knowledge and gut-microbe-epithelial interactions. Well, I think there are three major ones. One is that, as I mentioned, we have rudimentary knowledge and inventories of bioactive, microbedrive factors. But our understanding of the complexity and heterogeneity of gut-epithelial functions, particularly as they relate to microbial selection, assemblage, and region-specific interactions I think are incomplete. We also have an incomplete vetting and understanding of the above in the context of human biology and pathobiology. So at this point, I am going to broaden my comments to what I think are the challenges and needs. And this not only applies to studies of the gut epithelium, but to studies in general in the area of human microbiome research. And I grouped them into three categories. In the first category are human studies. So HMP1 was largely devoted to the study of humans. And I think that a lot of useful information came out of that project. But the limitations are the following. Most of the data that we have to date is observational or associative. And part of the reason for that is that people are really difficult to study. And they're different. You can't do much with them or to them. The other problem is that we often look at our microbiota data in the context of sort of archaic disease classifiers that clinicians often use to help them manage their patients. But these disease classifiers are based on symptoms and course in disease and not necessarily reflective of the kind of underlying pathophysiology. I think another limitation, and maybe this was a necessity, is that the HMP1 was really a technology-driven effort. And it was an important effort and helped us vet and develop some consensus in the way that we study gut microbes. But I think in doing so, the importance of biological drivers was minimized. And in part, they were addressed by the human demonstration projects. But I think we need many more human demonstration projects to move that forward. And associated with that is, we've taken a bottom-up approach. That is, we now have an immense amount of data that we are sorting through. And from that data, we put together hypotheses that we then look back onto the clinical situation and try to think if it's relevant. My view is that maybe that might be backwards and that we ought to really look at from the clinical situation and then from that develop various hypotheses that we will then develop or explore in clinical studies as well as experimental counterparts. I think there are major issues that I had brought up in the discussion yesterday, which are technical. We still don't have consensus on how to sample patients. I'll show you an example of that shortly. I think quality control is an issue. We don't have any well-accepted or standardized operating procedures. And I think that this really affects the interpretation of data. It's the old garbage-in, garbage-out story. And I think we have to pay much more attention to getting together and developing optimal ways to achieving a better upfront result. The other part of the technology is that I don't think that we've done a good job in vetting it. So I always tell my colleagues who show me the metatranscript or metagenomic data, I ask them, what does it mean? And how do you account for it? Do you really know that this data is true? And I think that we really haven't done a good job in actually vetting whether our interpretations of these omics data is correct. And in that regard, I think we need to do a better job at building a toolbox that can be more direct measures of microbial function. The third category, I lumped into experimental and data analysis. And here I think that I'm just going to echo some of the things that Sarkis and others have said. I think we really have to think about experimental and animal models as part of studies of the human microbiome. Just studying the human is not going to get you far enough. And I think the approach has to be where you have to do these two approaches in tandem. I think that the integration of large data sets is a problem, but also the fact that location is very important. For example, if you do a stool sample and you're looking at an inflammatory process that may be localized to a different part of the GI tract, how do you truly know that the alterations that you see in the stool are going to be reflective of what is happening at the local level? I think we need to think about or really strive to complete our inventories of microbial transcriptomes, proteomes, and metabolones because at the current point I think that this is a major bottleneck. Now let me give you some specific examples of the points that I tried to make. So I mentioned yesterday that inflammatory bowel diseases are described as two clinical phenotypes, one Crohn's disease, the other one called ulcerative colitis. They don't necessarily reflect the pathophysiology and the pathophysiology can be quite varied. So when we're trying to relate that to microbial data or host-microbe interactions, we may in some instances be comparing apples and oranges. But to add to that, you can look at what happens in patients with Crohn's disease. So this is data that came out of a study that was published many years ago, but reflects the fact that IBD, in this case Crohn's disease, is a progressive disease. And the natural histories among patients vary. So what you see in the early part of disease, which is mostly an inflammatory type, may not be what you see later in disease. And I think that that really affects the interpretation of your data. So if you did your sampling at T1, it's going to be very different from what happens in T2. And if you sample at T0, that is before the onset of disease, this is an ideal time point to be able to capture events before the disease happens. So you know a little bit of what cause and effect is. But the problem is that we have no way of predicting or identifying people who are at high risk for developing inflammatory bowel diseases. So most studies, in fact, I would say all studies pretty much have looked at post-inflammatory events. Now another challenge is this. So this is a lesion, an ulceration in a patient with Crohn's disease. And so what typifies these diseases is their topography. Ulcerative colitis only involves the colon. Always starts in the rectum. It's a mucosal disease. Crohn's disease, on the other hand, is a patchy disease. And it can involve any part of the GI tract. So if you look at this lesion, this is inflamed and ulcerated, but over here it's normal. So when we do a stool sample, what does it reflect? We really need to biopsy or sample around this lesion. Now if we sample, do we sample here, here, or in here? And how do we sample? Do we do a pinch biopsy or do we do a brushing? So none of these things have been truly resolved and there's no consensus. But they will affect the outcome of your data interpretation. So I may seem, having presented this, like a pessimist. But I can tell you, those people in my lab know that I am eternal optimist and I believe there's a solution to every problem. So one of them is that we were fortunately funded by the Human Microbiome Project to do one of the demonstration projects and we selected palchitis. This is a surgical procedure that's performed in patients with severe ulcerative colitis. They get their colon removed, which is curative. But for continents, what happens is the surgeons fashion a pouch that represents a pseudo rectum, which is anastomous to the anus so that they can have voluntary bowel movements. Now, half or more of these patients would develop a condition that looks very much like the original ulcerative colitis. And the other interesting thing is that if you do the same procedure in a patient that doesn't have IBD, somebody who has a familial form of colon cancer, very few of those patients develop palchitis. So this is something that we think is unique to ulcerative colitis and may recapitulate some of the pathophysiological events related to that disease. We know it's micro-dependent because the treatment of choice is antibiotics. Most of the patients will develop this condition within a year, which makes it an ideal project because you can do it prospectively. You can sequentially sample your patients unprepped and over time and be able to maybe capture events before they happen. And then patients serve as their own controls, which I think is very important to design in most human trials that look at the microbiome. Now, a lot of useful information has been gained from this study and Vince Young is going to present that later in the morning, so I'm not going to get into it. But even after looking at that data and trying to project out what it means, I think we're encumbered by the fact that we still have correlations and associations. So my argument is that we have to develop better animal models. And in this regard, I think that we propose a model that's been around a long time that fulfills a number of the criteria that recapitulates some of the events in the human pouchitis condition. So this is a blind loop that's surgically created so that this is the mainstream of the ilium and then off to the side is this pouch. And if you orient the motility this way, it empties, if you orient the motility this way, it fills. But over a period of days to weeks, this self-filling loop gets colonized and pretty much begins to look like a colon. As you can see, this looks somewhat like this. This looks like the ilium. And if you look at the microbiota, indeed they develop a colonic-like microbiota as we see in the human ileal pouches that clusters very closely to the sham-operated colon, whereas the self-empting clusters very close to the sham-operated ilium. And then if you look at how the microbiota from these pouches compare to the human pouch, you can see that up here in the green is the self-empting loop, which is away from the human samples here, whereas the self-filling is somewhere in between but closer to the human sample, suggesting that we're recapitulating some of the changes in human microbiota. Now, the other thing is, you have to ask why does pouchitis, if it reflects ulcerative colitis, occur in a small intestinal tissue? That's because that's what the pouch is made from. And we have to hypothesize that there are transformations of the small intestine to a large intestine, and this is actually seen in human pouches, but we also show that that is true in the mouse pouch. So this is just to show you the normal colon in the mouse, and then with the filling loop, you see that the histology with these elongate test tube-like crypts resembles that we see in the normal colon, whereas the emptying loop stays pretty much like the small intestine. And then the gene signatures are also very similar. That is the filling loop is very similar to the colon, whereas the emptying is close to the ilium. But we notice that even though we get colonization, that is insufficient by itself to cause inflammation. And so we added to this genetic variable where these IL-10 mice were subjected to the same procedure. You can see that with the genetic susceptibility, they develop inflammation, but if you appear in the self-filling loop, not in the self-empting loop. So the working model that we have from this mouse model is that this is three-legged stool representing the importance of colonic metaplasia. The development of a colonic microbiota may be dysbiosis and some form of genetic susceptibility. None of these are sufficient by themselves and you need to have the perfect storm in order for this event or palchitis and maybe ulcerative colitis to develop. So in moving forward, we're beginning to go back to our patients and beginning to look at the host side because looking at microbiota alone out of context with the host, I think we lost a lot of opportunity. But to look at the host side, we have to really understand that the gut mucosa is a complex multicellular tissue and that's shown by this picture here. This is the microbial world here and then there's a mucous layer in between and then this is us. But our mucosa is made up of many different types of cell types and if you just take a biopsy and you do, for example, gene expression, it's going to represent an admixture of cells. And I think through a variety of technologies, for example, laser capture, we can actually look at individual compartments and then through a systems biology approach determine how they fit together as a puzzle. And then there are other new technologies like the development of enteroids where we can develop fairly mature structures that we can then look at host-micro-relationships. So with that I'm going to end and I'd be happy to entertain any further discussion or questions. Thank you. Gene, thank you for a great overview and for, I think, clearly articulating some of the gaps, needs and challenges. I think we could have time for maybe one question while Balfour is coming up to the mic. Maybe I could just ask quickly in these enteroid models, are indigenous microbiota being retained as you develop these enteroids? No, so these enteroids are derived from stem cells that you can harvest. From the mouse it's fairly easy to do and the technology is now feasible even in humans. But they have to be established in sterile conditions. Sergeant North Carolina, Gene, great discussion points. We need a full afternoon, but I totally support your idea of host microbial interactions. We need to genotype all of these patients that we're getting the microbiota on and fully also support the mechanistic necessity for mechanism exploration and appropriately chosen mouse models, making sure that they're appropriate. And I love your self-enblinded quick question and that is on the chronicity of your HSP 70 knockout mouse with, I couldn't tell whether it's a single or several doses of DSS. Did that extend into the small intestine and could you comment quickly on the differences in the protective mechanisms and resiliency of the small intestine recovery versus the colon? Thanks for asking that. So actually what happens is that they, as you know, the IL-10 knockout mice are, the DSS is a typical distal colitis. What we see is that the colitis will extend like all sort of colitis up to the proximal colon, but it doesn't go into the ilium. Yeah, but it can. So in our GM knockout mice we actually get an ilitis in the susceptible host. And DSS certainly is pervasive throughout the intestine because you give it orally. So it's just that there is a particular propensity, but I was just wondering if it extended in that particular setting. It can in other genetically susceptible host. Thanks. Thank you. Our next speaker is Dr. Susan Erdman from Massachusetts Institute of Technology. Her talk will be Wound Healing Longevity, Harnessing the Microbe-Induced Hormonal and Immune Proficiency for Human Health.