 Thanks, Bill, for inviting me to this interesting meeting to share my data and some ideas. So as John Cleese used to say, now for something completely different. What I want to talk about now is not vast amounts of bacteria or vast taxonomic diversity or a whole lot of different cell types. I want to make a very focused talk on one cell type, the epithelia, and this intimate interaction here between the microbiota. So I'm talking about a very reductionistic, very simplistic dyad between one cell type and bacteria. And it turns out it's essentially one class of bacteria, which is completely different from what we've been talking about. But nonetheless, I think some aspects of reductionistic science does retain a lot of value in the analysis of the microbiota. Anyway, here's the obligatory slide, affects the microbiota on the gut. And we've been through this ad nauseum today. The pathogens, we'll hear more about that with the microbiota. Metabolic, nutritional energy utilization, vitamin synthesis, short chain fatty acids, all very well understood. Lots of great interest in adaptive immune regulation, induction of T cells by various subsets in the adaptive immune system. And innate immune regulation, dampening of immune responses, all classical responses that workers in microbiota and probiotics have been evaluating. In the area I want to just touch base on today is epithelial development and survival. It's well known under site of protective effects of signaling. Gene Chang talked about the heat shock proteins. But what I want to talk about is stimulation of barrier function and IEC, epithelial, intestinal epithelial cell restitution and proliferation. So some basic background, I'm sure this is common to most people here, or commonly known to most people here, is the epithelial in mammals is three dimensional. There's crips with the stem cells proliferating up to a transient amplifying compartment where they undergo differentiation and ultimately are shared to an apoptotic process. The differentiation can go into various cell types, absorptive, goblet cells that secrete mucins, endocrine and panacells. Anyhow, that's the normal process of the gut epithelia. However, germ-free studies over the years, even decades, have shown that the small intestinal crips show slower turnover of the epithelial cells with a criptavillus transit time doubling, indicating at least some enhancing role of the microbiota collectively in these processes. It's also been shown as markedly attenuated regenerative responses to colonic injury, implying their roles in restitution. So it brings up the question, so it's well known and no one here is going to argue that the microbiota can influence normal homeostasis in the gut, aside from the traditional innate immune responses. I'm not going to touch base at all today on the classic TLR or a nod protein. And everyone knows it's a very critical aspect, both of a innate immune and adaptive immune regulation, as well as underlying homeostasis. I'm trying to talk about a novel, extremely highly conserved pathway, which may work in parallel to those systems. But I'll go back to the question of how the normal microbiota mechanistically interacts with the epithelial is not well understood. It's kind of an obvious statement. And it's sort of a substatement in parallel, more specifically, is how the microbiota can influence epithelial growth and proliferation is also not well understood. So what I want to approach today is this concept about reactive oxygen species, ROS, and their influence on cell proliferation and differentiation. And build the case that this is a very ancient, highly conserved mechanism by which numerous path, numerous cell systems can stimulate growth and differentiation, as well as other signaling functions. But to go over some literature in the past, ROS, which are incompletely produced oxygen molecules, have potent signaling functions that are outside their more commonly conceptualized role as antimicrobial. And they've been involved in differentiation of proliferation processes in organisms as simple as social amoeba in the dictastelium, where superoxide signaling is necessary for its formation of the fruiting bodies once its contact with bacteria, its food source runs out. There's numerous papers showing plant and plant biology of transcriptional regulation of ROS by upregulation of various peroxidases. Proliferation differentiation is the root, is it migratory to the microbrit soil. Drosophila has a number of papers out showing differentiation and proliferation, particularly in the hematopoietic lymph gland of the fly. Two very, very recent papers in vertebrate systems in the, in Xenopus, showing a ROS signaling is involved in a tail regeneration and also a cell differentiation in mouse spermatogenesis. So just a little bit of background on the biochemistry. Reactive oxygen species traditionally been conceptualized as highly reactive deleterious molecules from the incomplete reduction of molecular oxygen. Hydroxy-radical, hypochlorous acid and peroxy nitrate indeed are highly damaging to macromolecules. They can cause a lot of damage associated with the aging response, except when they're harnessed as an antimicrobial action which occurs in the phagocytic vacuoles of phagocytes. But this is the traditional role. However, it's very recently appreciated that there's a lot of signaling roles for the non-radical ROS, such as hydrogen peroxide and nitric oxide, which have potent roles in transiently and non-permanently modifying enzymes. And this brings up this concept of redox regulation of enzymes. It's a subclass of enzymes that are defined by a cysteine residue in the active site that exists as a thylate anion at low pKa. And in the presence of H202, it can undergo rapid and transient modifications of sulfenic acid and disulfide. This is a form of oxygen stress. And it's very rapid and very reversible. It can be highly localized. Known target enzymes include numerous enzymes involved in the ubiquitin-like protein processing, including sumo and net-8, dual-specificity kinases involved in map kinase signaling, low-molecular PTPases and cell motility, and some pathways that are actually involved in redox sensing itself. So the ROS generated is not exogenous. It's deliberately generated by a class of enzymes called the NOx enzymes. This brings up why the hell would all these cells and organisms have enzymes that deliberately produce ROS? It's generally found for these signaling functions. The original member is what's now called NOx2, originally called GP91-Fox, which is the classic respiratory burst enzyme in million phagocytes. And the old-timers here will recognize the formal peptide receptor-stimulated ROS-oxidant burst. It's one of the canonical reactions of experimental pathology described back in the 50s or 60s. But anyhow, subsequent work has shown these NOxs are expressed in... There's five forms expressed in the human in many, many cells, including neurons, epithelia and endothelia that are non-phagocytic. And they're expressed all through the... All through metazoans, including plants and other animals. In each case, plants and animals, in each case it's been implicated in response to microbes, indicating it's a very highly conserved fundamental pathway where multicellular organisms respond to bacteria, including plants which have numerous defensive responses when they're invaded. In C. elegans, defensive responses to a duox-like molecule in C. elegans. Drosophila, there's both a duox and a NOx. We'll spend some time on this with the Drosophila system. We've mentioned mammalian professional phagocytes. And the NOx1, which is present in mammalian barrier epithelia, which is obviously relevant to our discussion today. The other aspect and background I want to bring up is this concept of the formal peptide receptor pathway, which again was characterized in phagocytes as a response to virtually all bacteria. It's what causes neutrophils to migrate towards the formal peptide. It's essentially a PAMP. It's a modified translation product from all bacteria. And what brought this to the forefront is when we and our collaborators discovered these formal peptide receptors were expressed actually on the epithelia of the gut and other epithelial tissues. In a series of papers over the years we characterized this pathway, whereas the FPRs can monitor, detect formal peptides, activate NOx family members, produce ROS, cause inactivation of various target enzymes and subsequent downstream effects, including effects on motility, proliferation, and immune suppression. I'd like to discuss aspects on epithelial movement and proliferation further. So this starts with our earlier observations showing that in vitro and in vivo the commensal bacteria can stimulate ROS generation. These experiments were facilitated by the development of these specific dyes that can put on the cells or non-toxically injected into mice so that we can look at the tissues. But he takes cells, you can add, here we're using lactobacilli. Now lactobacilli will be a theme of this talk, which I'll come back and circle around and discuss. But lactobacilli causes a rapid and transient induction of ROS in vitro, and also in vivo when we gavage mice either by gavage or transrectal installation. This is actually a small bowel villi confocal within 60 minutes of gavage. With lactobacilli, you can see the activation of the ROS. One of the most interesting aspects of this work was found is this phenomenon is restricted to a subset of bacteria, predominant lactobacilli and also bifidobacterium, numerous other bacteria, B-theta E. coli, and numerous others fail to show this response. So it's a true biologic response that's limited to aspects of the, specific members of the microbiota. We will take isolate germ sequel contents from mice, use this in vitro assay and show a similar effect, whereas from germ-free animals the sequel contents at a similar density or turbidity did not have the ability to stimulate ROS. And in vivo similarly by gavaging these various strains, you can see that the lactobacilli is by far the most potent inducer of this response. This response is dependent on NOx1. This is very recent unpublished data by Ronald Jones, who's in the audience here. These are just different areas of tissues, the colon and the small bowel, showing the activation of ROS by lactobacillus rhamnosus. And it's abolished in both cases in animals that have a epithelial-specific knockout of the NOx1, ROS-producing enzyme. The other thing we're able to do, this is a workup by Ashfa Colomels in my lab, is that these effects are enhanced around the wound edge, which is a common phenomenon. This is an in vitro assay, essentially a scratch wound. We can take a cultured monolayer, scrape them, use the dye and stimulate them either with bacteria or pure formal peptide and show you have activation of ROS, especially on the edge. And here's a novel system utilizing a veterinary endoscope where we can insert it into the distal colon and use a biopsy to inflict wounds that we can come back and evaluate. This is a method pioneered by Thad Steppenback. With our ROS dyes, we're able to show strong up-regulation of ROS. It's even more enhanced along the wound edge, which will be relevant in a moment. Non-ROS-producing bacteria or stimulating bacteria such as E. coli will not do this. And very importantly, the ROS response is lost both in the NOx1 null animals and the FPR null animals. And also importantly, the response is preserved in NOx2 null animals indicating this is not coming from phagocytes nor in mid-88 null animals. You get perfectly good response indicating this is not going to at least the most of the TLR pathway. Now here's another body of work that's very similar. This is Drosophila. So I mentioned Drosophila is a great system of work for cell reasons. One is has a reduced genome. So there's only two NOx producing it. And two NOx is a D-NOx and a D-DUox. So it's reduced on the host side. And also they have a wonderfully simple microbiota that's 10 to 20 organisms depending on how you culture them. And given their size, all these organisms are aerobic and they can generally be cultured. So this is a data, again, by Ronald Jones. He was able to generate azenic flies. Again, a simple procedure by taking eggs, de-coronating them in fluorox essentially and putting them in autoclave media and monocolonizing them with various bacteria. And he can show that within minutes of certain bacteria, lactobacillus, you see the gut lighting up and here at higher power individually on the epithelial cells of the fly. Even more remarkably, this is a, in first instar larva. These are much smaller ones as they hatch. So here you can take the larva, have them hatch into media that is monocolonized with specific bacteria. And you can see the lactobacillus again stimulates the epithelial cells using the dye in an alternate technique with a transgenic fly line and with a redox responsive reporter gene. What's remarkable about this system, it's a great system for looking at. It's a multi-cellular organism that you can look at. It's initial colonization with its microboat essentially in real time instantly as they hatch. But what's remarkable here is of the dozens of bacteria we tested, only the fly specific lactobacillum, the lactobacillus planterum was able to induce ROS. And that's, we've had previous talks talking about some specific properties about the lactobacillus. I think it's a very fascinating area. What's specific about this taxa that mediates these effects across many, many different assays? So similarly, just a similar type data. This is fly gut showing the ROS induction. We're able to utilize fly genetics to knock down the two ROS producing enzymes, D-NOx, showing it's lost, D-duox, which is said or generally thought to have more of a defensive function. You do not see the loss. This is highly consistent, perfectly consistent with what's seen in the mice. And now to change the subject, going from the ROS production and some of the consequences of ROS production, I'm going to talk a little bit about cellular motility. As I mentioned, ROS production and formal peptide receptors are involved in the motility of phagocytes. And this property seems to extend also to epithelial cells. It's well known that epithelial cells are in a wound. They differentiate and move along focal adhesions by the action of focal adhesion kinase, which is held in its inactive state by phosphotyrosine phosphatases. Physiologically, ROS is produced by NOx, which is in physical proximity to these PTPases, causes inactivation of fact, phosphorylation formation of actin bundles, and the initiation of motility. It's a classic cell biologic phenomenon. What we were able to show, we sought to see whether this phenomenon was applied to bacterial induced ROS. Not to get into the details of the biochemistry, but we can use an ethyl maliumide, a probe that will bind to the phyl anionic forms, enables a nice pull down assay when it's oxidized by bacteria that's lost, as well as enzymatic activity. When the enzymatic activity of PTPase is gone, fact is activated. This is the in vitro and in vivo data showing on a wound, on a monolayer, when they're treated with lactobacillus, you see increase of the phospho fact, both overall and specifically along the wound edge. When you co-localize with a phylloidin stain, that's actin bundles, you can see how they co-localize and actually see the nidus formation, the yellow, where the actin bundles are actually sticking and emanating out of the areas of the focal adhesion kinase. So lactobacillus actually stimulates focal adhesion formation. In vivo, on a wound bed, the coars are reversed, and the faking fact is activated at the basal lateral aspect of the wound bed by rhamnosus. Functionally, we're able to show on a wound bed assay, these are cells by photovidium microscopy, that lactobacillus actually accelerates the pseudopodia extension and the healing of the wound that can be quantified in a velocity calculated in vitro. And in vivo utilizing the method of stabbing back by inflicting wounds with the endoscope and then measuring over a daily basis and then utilizing an image analysis software when it can calculate wound rates, show that lactobacillus does indeed induce wound healing. It's similar to what we saw earlier with the skin, although I must admit this is in contact with lactobacillus through gavage. It's not a systemic effect. And importantly, this wound healing effect is lost in the FPR null animals and the NOx1 null animals. We can also show in intact epithelial, the barrier function is measured by fluorescent dextrances, is increased by lactobacilli of this type as other workers have shown. More in the proliferative aspects, Mapkinase urk is stimulated by lactobacillus and formulated peptide as shown in vitro. And again, the effect is lost in FPR and NOx1, the signaling components for the bacteria, but not in mid-88. And then also the pure proliferation is induced by lactobacilli. This is two different experiments or assays for proliferation in the small bowel of the mouse, looking at phosphohistone 3 or EDU incorporation. This is data similar to what the lab described a year or so ago that lactobacillus causes a marked stimulation of growth within the transient amplifying compartment of the small bowel. In this case, what we're able to show is this effect is lost, pretty much abolished or absolutely abolished, in the NOx1 NO animals and also in other data in the FPR NO once, indicating that they're involved in this pro-proliferative response. And then to bring the whole thing full and around, in the fly system, it's a two-dimensional sheet, not three-dimensions. You have the enterocytes, stem cells, and the equivalent of the transient amplifying compartment. Under normal conditions, you can use EDU to label them. Under germ-free conditions, it's greatly suppressed, EDU incorporation can be brought to supernormal levels by the addition of lactobacillus within four hours, and that's abolished by the addition of a redox sync. And then finally, the genetic experiments, the NOx knockdown, abolishes the proliferative effect in the flies, whereas the duox knockdown has no effect. So the conclusion, and this is from the poster of Ronald Jones, it's number 20, it's still out there, is NOx dependent generation of physiologic levels of ROS by lactobacilli and likely other bacteria. That's an interesting point there. It's a novel signaling mechanism for transducing bacterial signals in the host regulatory events that mediate intestinal homeostasis proliferation and restitution. So the future challenge is, what are some novel, raw sensitive proteins and pathways? We've happened to come across three or four of them that had been described in the non-microbial physiology in the past, including a FAC activation and the MAP kinase signaling. There are proteomic approaches available utilizing these redox sensitive probes that can enable us to evaluate the whole proteome that's stimulated by ROS produced by bacteria. So that's an area of future development. What is the entire pattern of our collection of proteins that can be stimulated in a sense? It's a correlation with innate immunity and PRR signaling. There's a lot of crosstalk here. FPRs are considered an immune receptor. Certain TLRs have been described to induce ROS under certain circumstances, and ROS production is a known inducer for some of the inflammasome processes. So there's some crosstalk here getting into inflammatory and disease-related biology. Another fascinating question is correlation with microbial determinants and bacterial taxonomy. What is it about these specific bacteria? If it's lactobacilli, is it adhesive property? Is it a single-component individual component? A red pulp has shown the P40 and P60, I believe, components of lactobacilli that seem to mediate beneficial effects, and I would like to look at that for stimulating ROS properties. And further bacterial taxonomies, what other bacteria are able to mediate these effects? Lactobacilli are very interesting because they colonize, as we heard today, the mammalian got very rapidly. They break down milk oligosaccharides, and at least in the mouse, they form a very rapid biofilm. So they've obviously co-evolved with mammals over millennia, and likewise in flies, they break down some of the oligosaccharides in the fruit that the fruit lives in. So there's an evolutionary sense to the idea that certain bacteria have co-evolved by virtue of utilizing the energy source that the young animal, whether it's invertebrate or vertebrate, utilizes. And finally, just the idea here that we're showing that the bacteria can control epithelial movements and proliferation. What is its role in normal gut development over time in, say, neonates or very young animals in wound healing or the roles in probiotics that we and others have shown can decrease intestinal permeability and affect a lot of systemic biology and potentially a role in oncogenesis. So I'll basically stop there. I'm going to ask comments into some gaps. This is more of a technical talk, so I don't really want to get into technical issues. But the one concept I want to bring up is epithelial cell biology is a host microbial system. We heard yesterday the appropriate alarm that a lot of our immune systems in these mouse models have to be taken with a grain of salt realizing the alterations in the microbiota can affect the adaptive immunity in the whole organism. And from the perspective of an epithelial cell biologist, one has to accept that epithelials don't exist in a dish by themselves. They are chronically-tonically in contact with bacteria of different densities, different taxonotic compositions. So future experiments looking at epithelial cell biology really have to keep in mind the fact that they evolved or designed to be exposed to the microbiota. So I think that's an important gap that needs to be perceived on. The other thing, just more technically, I think some of this work does show some of the advantages of model system work in flies. I know there's other workers that utilize zebrafish. It is a nice vertebrate model. C. elegans can be used for some of these simple systems and others. I think it'd be an interesting area to develop or future meetings or future symposia for people utilizing some of these experimental systems, which can be usefully reductionistic for looking at aspects of host microbiota interactions. This sort of brings me to the final comment. Comparative metagenomics. Obviously a fly only has a couple dozen organisms in its microbiota, but they obviously had some critical roles in controlling proliferation and nutrition, as we've heard. So some of the comparisons, these much more simple microbiota, might be informative in bringing sense to these very large data sets we have with the mammalian systems. So I think I'll stop there. Acknowledge workers in my lab, Ashfaq Alam, Huisha Wu, Jeff McKinney, Phil Swanson, my collaborator, Ronald Jones, who did all the fly work, and Osman Nusrat, who helped us with the cell biology, and of course for support from the NIH. Thank you very much. Thank you, Andy, and thank you for minding the time so well. That does leave us time for a couple of questions. And while people are making their way to the microphone, I'll start off, I think. Have you had a chance to do any kind of characterization studies of the commensal factors that are activating NOx1? Yeah, the little smidging of data we have is we have some mutants in the lactobacilli that affect some of the S proteins in the cell wall, which are said to affect adhesion. And we're in the process of looking at that now. But preliminarily, it does look that defective adhesion by the lactobacilli reduces a lot of these responses. But that's a whole microbiologic frontier right there in and of itself. Do you know if the lactobacilli are activating any specific toll receptors? Well, they're not activating any of the... the mid-88... the responses aren't going through mid-88. And so that's a hard question to ask, because if we're using... over the years, you can get conditions where lactobacilli will activate traditional pro-inflammatory signaling in epithelial cells cultured. But if you have a fully polarized, true gut epithelial model, they do not respond to lactobacilli at all. You can get ERC activation, which is some of the prompt of this whole business, looking at ROS, but you don't see junk, you don't see NFCAPB, you don't see P38 or others. But something or ACT will come up and some others, and that sort of steers in this pro-proliferation away from the traditional inflammatory models. Senors. Do these various pathways operate under a physiological hypoxia? Physi... elaborate on that a bit? I mean, especially in IBD, there's a state of hypoxia in the gut epithelial layer. And I'm just wondering if these pathways will operate under that condition. Well, it certainly will operate during oral gavage in a mouse when we dissect later. So that's as physiological as we can get. In the flies, I mentioned, there is no physiological hypoxia which is a very valid concern with the in vitro studies because none of those are going to be truly, for various reasons, they're just not going to model aspects of cell biology or host biology like what you're saying there. Thanks. Okay, thank you very much. Thank you. So the next speaker is Peter Tanba from Howard University and the title of this talk is Moving Towards a Metagenomic Basis of Therapeutics.