My name is Dan Littman. I’m a professor of Molecular Immunology at the Skirball Institute, which is part of New York University School of Medicine. And I’m also an Investigator of the Howard Hughes Medical Institute. What I’m going to tell you about is how the microbiota, as well as other components of the environment, influence the immune system at barrier surfaces. I’m going to focus mostly on the intestine, which is the area that’s been best studied by many groups around the world during in the last decade. And I’ll tell you, in my first part of the talk, about the different kinds of cells that are involved, as well as some of the signals that are involved, particularly in the functions of lymphocytes, in lymphocytes that are both within the innate immune system and the adaptive immune system. The adaptive immune system consists of B lymphocytes and T lymphocytes that differentiate from common lymphoid progenitors. And these are adaptive because they have rearranging genes that give rise to T cell receptors or antibody receptors on the surface of these cells, so that each cell has a clonally restricted type of receptor. The innate cells… lymphoid cells also differentiate from a common lymphoid progenitor, but they have fixed receptors, and they are basically hardwired to respond to various cues that are presented to them, be they cytokines or be they some type of danger, antigens that are presented to them. So, I’ll start out talking about the T cells, and in the second part of this presentation I will tell you a little bit about some of the innate lymphoid cells. T lymphocytes develop in the thymus from common progenitors that… that enter the thymus as so-called double negative cells. And the double negative cells are so called because they don’t express on their cell surface the molecules CD4 and CD8. As they undergo development, these cells can take one of two lineages. One lineage is to become gamma delta T cells, meaning they have receptors encoded by the gamma and delta genes. Or they become alpha beta T cells. The alpha beta T cells typically express both CD4 and CD8 on the surface as they become double positive cells. And these cells develop, if they have appropriately rearranged receptor genes that give rise to the protein on the surface, a heterodimer of the alpha and beta chains. The vast majority of these cells undergo cell death, because for a cell to develop in the thymus, it needs to have a receptor that interacts with self MHC proteins, major histocompatibility complex proteins, that have peptides presented to the T cell receptor. So, since most of the cells do not have an appropriate receptor, they undergo cell death. A few of the cells also have receptors that interact with very high affinity with self-antigen. And these are potentially damaging cells that can lead to autoimmunity, and those are also eliminated through a process called negative selection. And then the few cells that make it through this gauntlet undergo positive selection, and most of them become either CD4-positive cells or CD8-positive cells. The CD4-positive cells are those that are selected on MHC class II molecules, and they are typically helper cells. And I’ll talk a great deal about this throughout the rest of my presentation. The CD8-positive cells are selected on MHC class I, and they mostly become cytotoxic or killer T cells. There’s another type of CD4 cell that’s also selected on MHC class II. Typically, these are cells that have a higher affinity receptor. And these cells up regulate the transcription factor Foxp3 and become regulatory T cells. So, these are thymically derived regulatory T cells, which are essential to maintain tolerance in the periphery and prevent autoimmune activation of the other types of cells of the immune system. I’ll tell you a little bit about another type of regulatory T cell that arises in the periphery, the so-called induced Treg cells, in… a little bit in this presentation and a lot more in the second part of my talk. Once these cells develop the thymus, they are exported into the periphery. And most of the classical alpha beta T cells, the CD4 cells and the CD8 cells go to secondary lymphoid organs, where they are naive T cells awaiting to be activated by immune signals, potentially from invading microorganisms, and following activation of innate immunity. But there are also cells, such as the gamma delta T cells, as well as some subsets of alpha beta T cells, that go directly to peripheral organs: into skin, the epithelium in the intestine, as well as the lamina propria in the intestine, as well as the female reproductive tract, or the lung. And these… these are cells that are very much like innate lymphoid cells, in that they can often be activated very quickly to deliver the cytokine load that they have. But the more conventional cells from the lymphoid organs, once they are activated by antigen in those organs, can migrate out to the very same sites in the periphery, into these different… different tissues. And once these cells migrate to the different tissues, they can stay there and continue to replenish themselves. And these become tissue-resident memory T cells. And they consist not only of the T lymphocytes that I just mentioned, but also of innate lymphoid cells that I’ll tell you more about, and also macrophages. In particular, there are different populations of macrophages, some of which arise very early during fetal development, that establish themselves into tissues and then reside in those tissues for the life of the organism, continuing to replenish themselves. So, these are to be distinguished from the other types of cells that are circulating between the blood and the lymph and the secondary lymphoid organs. And that is a distinction that one should keep in mind. So, these are many different tissues that now harbor these tissue-resident cells. An example is shown here from work of Daniel Mucida, in which he described T lymphocytes that… that established themselves in the epithelium of the intestine, in this case, the small intestine. And you can see the tracings of these cells as they traffic through the epithelium. They basically undergo a flossing-like movement in which they are detecting potentially harmful pathogens as well as any kind of damage to the tissue, and then make the cytokines and growth factors to repair the tissues, or rid the organism of the potential pathogen. I’m going to focus mostly on the CD4-positive T cells, and most of these cells are helper T cells, because they help the cytotoxic T cells to undergo their functions. They also help B lymphocytes to make antibodies. But it’s… about 30 years ago, Tim Mosmann and Bob Coffman, at the DNAX Institute at that time, first described different properties of CD4-positive T cells in that they could make different types of cytokines. What they found was one subset of cells made the cytokine interferon gamma. And they and others then found that these cells are critical for killing a variety of intracellular microbes — bacteria, viruses, protozoa — are controlled by these T helper 1 cells. These cells express the transcription factor T-bet, which is required for their differentiation. The other cell type that they identified secreted interleukin-4 and interleukin-5, and later was shown that they also make IL-13. And these are critical for controlling infection with helminths, or parasitic worms. And on the other hand, these cells are also very important in allergy and in asthma, and need to be controlled. The Th1 cells were initially thought to be the key cells involved in autoimmune disease. But about a decade ago, a third type of differentiated T cell was described, called the Th17 cell. And these cells are so-called because they make the cytokines interleukin-17A and interleukin-17F. They also make interleukin-22. And it was found that these are actually the cells that are most often involved in autoimmune inflammation. And these are cells that are normally needed to kill extracellular bacteria and fungi at mucosal surfaces. They’re very important for repairing damage to mucosal tissues. And in a number of different models for autoimmunity, they have been found to be the critical cells. But more important, they have been found to be critical cells in autoimmunity in human. I’m going to tell you a lot about these. First of all, just as a way of background, the IL-17 cytokines are very important for inducing chemoattractant cytokines, chemokines that attract neutrophils to the site of the secretion. They’re also involved in tissue remodeling. IL-17 induces matrix metalloproteinases, as well as VEGF, which leads to angiogenesis in a variety of tissues. Interleukin-22, on the other hand, is more of a cytokine that leads to proliferation of epithelial cells, and protects the barriers from damage. There’s another, fourth cell type that I want to describe here, and I mentioned it briefly already. That is the induced regulatory T cell, which, like the one that’s made in the thymus, also expresses FOXP3. But in the periphery, these are cells that are induced by combinations of cytokines, particularly TGF-beta and retinoic acid, as well as interleukin-2. And you’ll note that the Th17 cells can also rely on TGF-beta for their differentiation. And I’m going to concentrate on telling you a little bit about the requirements for the differentiation of these cells into… in these different directions. When a T lymphocyte is activated, it requires two signals through… in order to proliferate and produce their cytokines. One of the signals of course comes from the signaling pathway linked to the T cell antigen receptor, which interacts with MHC and… either class I or class II MHC and peptide. But the second signal is mediated through CD28, which is called a costimulatory molecule. It interacts with ligands on antigen-presenting cells, on specialized antigen-presenting cells, particularly dendritic cells. And only when these two signals are integrated will the cell, now, become activated and proliferate. But a third signal is needed for the differentiation. And that is a signal provided by cytokines that are made, also, by these antigen-presenting cells most of the time. And these cytokines signal through a variety of different receptor subsets in order to provide the cell with the… with the function that it’s going to adopt. An example of this is shown here, in which I show the critical cytokines involved in the differentiation of Th1 cells and of Th17 cells. And these are the cytokines interleukin-12 and interleukin-23. IL-12 and IL-23 share a subunit, the p40 subunit shown here. And they also share a receptor, the IL-12 receptor beta-1, you can see is present as a receptor for both cytokines. But then they also have unique subunits — p35 for IL-12 and p19 for interleukin-23 — as well as unique receptor polypeptides, which link them up to the signaling pathways downstream. Much of the early confusion about Th1 cells was because of these shared components, of p40 and the IL-12 receptor beta-1, and some of the functions for IL-23 were ascribed to IL-12 at the time. But there’s been a lot more clarity in the last few years, with the discovery of IL-23. And what we now know is that these receptors both signal through the JAK-STAT pathway of signaling… of signaling molecules. The JAKs are cytoplasmic tyrosine kinases that are engaged by the different receptors. And they transphosphorylate to become activated, and then phosphorylate different types of STAT proteins, which are transcription factors, which, when they are tyrosine phosphorylated, form dimers that then translocate to the nucleus and activate a variety of sets of genes. In the case of IL-12, it activates the STAT4 transcription factor. In the case of IL-23, it activates the STAT3 transcription factor. And many of the other cytokines that I’ll tell you about, such as interleukin-6, also activate STAT3, but each of these receptors that utilizes STAT3 also has distinct signaling components to target particular genes. STAT4 can also, under some circumstances, be activated by IL-23. And that is when IL-23 is engaged in pathogenic processes in vivo. And I’ll tell you a little bit about the IL-23 function in the next… in the next few slides. The key experiment that introduced the concept of Th17 cells and showed the importance of interleukin-23 was from the laboratory of Dan Cua at DNAX in 2003. What they did was to use a model that’s widely used for multiple sclerosis, called experimental autoimmune encephalomyelitis. And this is a model that I’ll be talking about throughout my presentation, because it’s often used because it’s fairly rapid and it’s fairly robust. In this kind of a model, a myelin protein is injected into mice along with adjuvant. And typically, within about two weeks, the animals began to develop paralysis. And as you can see over here, in wild type mice the paralysis develops as expected. And in mice that are deficient for p40, which, as you recall, is absent in… in… leads to an absence of both interleukin-12 and interleukin-23, you can see these animals are protected. But the surprise at the time was that mice lacking just IL-12, that were deficient for p35, not only developed disease, but they actually had even more severe disease than the wild type. Whereas mice deficient for p19 — that… what was then the newly discovered component of interleukin-23 — were completely protected. So, that then led to the important concept of the target of p19 as being the Th17 cell. And these cells are critical for barrier defenses against a variety of different bacteria and fungi, fungi including Candida albicans. But the flip side of this is that these cells can also be highly pathogenic through the production of their variety of cytokines. And under inflammatory conditions, they can now produce not only IL-17 and IL-22, but they can also make interferon gamma. And these cells have been validated to be very important in many human diseases, particularly psoriasis. In psoriasis, antibodies against interleukin-17A are very effective in therapy. Also, a variety of different arthritides, psoriatic arthritis and ankylosing spondylitis in particular, can be treated by blockade of interleukin-17 and interleukin-23. And then there are also some other… there are some other suggestions that multiple sclerosis, as well as a variety of inflammatory bowel diseases, are Th17-mediated diseases. For IBD in particular, it is known that polymorphisms in the Th17 signaling pathway can contribute to the disease, again adding further argument for a role for these kinds of cells in the disease. There’s also some evidence from animal models that Th17 cells in the mother can influence the development of the fetal brain, if they are expressed in very high levels and can cross the placenta. This is only an animal model that has been looked at, but I will discuss this in the second part of my presentation. At the center of the Th17 differentiation process is a transcription factor, ROR gamma t. And ROR gamma t is encoded by this locus, Rorc, in which two different isoforms can be ex… can be transcribed, depending on the promoter that’s used. And the longer form is expressed quite broadly, and it’s expressed in a circadian manner. But the shorter form of ROR gamma t is expressed exclusively in lymphoid lineage cells. And these include the cells that develop in the thymus, the double positive thymocytes that require ROR gamma t for their survival, as well as lymph nodes and Peyer’s patches, secondary lymphoid organs that develop in the fetus and that require the lymphoid tissue inducer cells that are dependent on ROR gamma t. In this slide, I show a crystal structure of ROR gamma t, of the ligand binding domain, which regulates its function. So, this is a nuclear receptor, very similar to estrogen receptor and glucocorticoid receptor. And it is thought that it’s regulated by ligand, but the precise ligand has yet to be defined. What we know is that molecules in the cholesterol biosynthetic pathway are very effective at regulating ROR gamma t function. But we don’t yet have strong genetic data to tell us which of these intermediates are important in vivo, and in particular, in the differentiation of the different cell types, whether there may be different ligands that are involved. Now, in… beyond the function and development, the transcription factor, of course, is required for Th17 cell differentiation. It’s also plays… playing a role in the differentiation of the induced regulatory T cells found in the intestine, in particular in response to microbiota. And I’ll talk more about that in the second part of my presentation. Also, there are gamma delta T cells that are specialized to make IL-17, and that’s also dependent on ROR gamma t. And then there are the innate lymphoid cells, and these include the lymphoid tissue inducer cells, both those that are involved early in the fetus, in lymphoid development, but also those that appear… that develop postnatally and that are involved in some of the tertiary lymphoid tissues. I’ll talk a bit more about the type III innate lymphoid cells a little bit later. But I want to come back to the Th17 cells. And this experiment that we did a bit more than a decade ago showed the importance of ROR gamma t in the EAE model. So, you can see here that animals deficient for expression of ROR gamma and ROR gamma t are highly protected from EAE. And you can see that there are very few IL-17 producing cells, shown by the FACS analysis, over here. These are cells in the central nervous system. On the other hand, in the wild type mice, you see not only that there are IL-17-producing cells but also cells that make both interferon gamma and interleukin-17. And it turns out these are very important cells, because these are those cells that are found in pathological situations, in which there is tissue destruction and autoimmunity. I will discuss two pieces of evidence that suggest a critical role for interleukin-23 in the generation of these cells that produce both interferon gamma and interleukin-17. One of the experiments is from an in vitro model for EAE, in which it is possible to differentiate cells into Th17 in vitro, and if these cells are specific for a myelin protein, then they can be injected into mice. And within two weeks the animals get EAE. So, people typically use transgenic mice in which the transgene for the T cell receptor is for a T cell receptor that recognizes a myelin protein. And typically, most people use, in vitro, interleukin-6 and TGF-beta to differentiate cells into Th17 cells that make interleukin-17 and interleukin-22. But it turned out that under these conditions these cells did not induce EAE. On the other hand, when interleukin-23 was included, it was possible, now, to get EAE. And it was even possible to do so in the absence of TGF-beta, just by including interleukin-1 beta instead of TGF-beta. In that case, again, EAE could be induced. And so the interleukin-23 molecule is critical in this process. And this is a very elegant experiment that was done by Brigitta Stockinger’s laboratory in England, in which they showed the importance of IL-23 in the generation of these Th17 cells that make interferon gamma. What she did was a fate mapping experiment, in which the yellow fluorescent protein is knocked into a ubiquitous locus, but it’s only expressed when a transcriptional stop signal is excised by the action of Cre recombinase. So, she bred these mice to animals in which the Cre recombinase was knocked into the IL-17a locus, so only those cells that make IL-17a will have the capacity to then express YFP. So, upon expression of Cre, the stop signal is excised. YFP is expressed for the life of that cell, even after the cell stops making IL-17. So, what the group then did was to gate on just those YFP-positive CD4 cells in the model of EAE, looking now at what happens. And of course, in the absence of interleukin-23, there is no EAE. But they were able to now look at these YFP-positive cells and see what their phenotype was. And you can see here in wild type mice, many of these cells, now, express interferon gamma, or a combination of interferon gamma and interleukin-17A. But these cells are absent in the absence of interleukin-23. So, that really provided the critical piece of evidence that these double-producing cells are important in the pathogenesis in this model. But there’s evidence that it’s important in pathogenesis in other models as well. So then, what is the role of TGF-beta? Because, as I showed you from the in vitro experiment, it seemed like TGF-beta was not necessarily needed to be able to induce EAE. But on the other hand, if… when people looked in animals in which the TGF-beta receptor was ablated, they saw that there was no EAE, compared to the wild type mice, here. So, how could one explain this? Well, a recent paper by Zhang et al has begun to shed some light on what TGF-beta is doing during differentiation of Th17 cells. And typically, when interleukin-6 signals by itself, through phospho-STAT3, there is no expression of ROR gamma t and no IL-17 that is made. But that appears to be because the… one of the targets of the TGF-beta signaling pathway, SMAD4, recruits a co-repressor complex that includes the SKI molecule, which itself brings in the histone… histone deacetylases. And the histone deacetylases basically shut down chromatin at the ROR gamma t locus. But when TGF-beta is applied, that now leads to a degradation of SKI, of SKI, and basically the relief of the histone deacetylase function, and activation of transcription of ROR gamma t. And now interleukin-17 and other cytokines can be produced. We don’t really know yet whether some of the other targets of the TGF-beta signaling pathway, like phosphorylation of SMAD2 and 3 have some positive effects here, but what’s clear is that the TGF-beta relieves this negative function. And indeed, what you can see here, in this very nice experiment… if, in the TGF-beta receptor knockout mouse, one introduces also a mutation, a deletion of the SMAD4 locus, now EAE can be restored because Th17 cells can now differentiate in the absence of TGF-beta. So it appears, then, that, in least in vivo, TGF-beta is also important in autoimmunity, although in… even though it doesn’t appear to be necessary in the in vitro models. So, what I’ve just shown you suggests, then, that there are two different types of Th17 cells. Those that can differentiate in the absence of interleukin-23, and that can make interleukin-17A and F, and IL-22, and these we can call homeostatic or non-pathogenic Th17 cells. They typically are induced by microbiota, and they’re found at barrier surfaces. On the other hand, in various cases of inflammation, which can be found in different types of tissues, interleukin-23, along with IL-1-beta, but at least in some cases probably aided by TGF-beta, leads to the differentiation of these cells that can make not only the Th17 cytokines but also Th1-like cytokines, like interferon gamma, and that can contribute, now, to disease. And these kinds of pathogenic Th17 cells have also called… been called Th1* cells in human. And I’ll summarize for you what we currently know about these Th17 cells, be they non-pathogenic or pathogenic. So, the homeostatic cells are typically induced by the commensal microbiota and protect the mucosal barriers. And they produce not only IL-17a and f and IL-22, but also interleukin-10, which is an anti-inflammatory cytokine. But the pathogenic Th17 cells are induced by selected microbial pathogens, or what we call pathobionts, which can live in our bodies under normal circumstances without causing disease, but then can be stimulated to cause disease in some circumstances. They participate in the autoimmune diseases, dependent on interleukin-23. And in addition to the Th17 cytokines, they make these other cytokines, particularly interferon gamma. And the human equivalent was described by Federica Sallusto. These are cells in the circulation that produce interferon gamma, but they also express target genes for both Th1 and Th17 transcription factors, such as CXCR3 and CCR6, which are chemokine receptors there are targets of Tbet and ROR gamma t, respectively, the Th1 and Th17-specifying transcription factors. So, there are many mutations that have been now described in humans that give us some clues about the Th17 pathways. There are individuals who have chronic cutaneous candidiasis, and oftentimes there are mutations in interleukin-17, interleukin-17 receptor, as well as the signaling components that have been identified. And then there are a few patients who have been described who have disseminated mycobacterial infections after immunization with BCG, which is used as a vaccine for tuberculosis. And it was found that ROR gamma t mutations can account for this, in which there is loss of both the Th1* cells and Th17 cells, even though there’s no effect on Th1 cells that can serve an anti-viral function. And some of these mutations are depicted here in this slide. The STAT3 mutations, both gain-of-function and loss-of-function, have been described. And also a gain-of-function mutation in STAT1, which blocks the differentiation of Th17 cells while promoting Th1 cell differentiation. Those kinds of mutations can also account for some of the cases of candidiasis. Mutations in IL-17… IL-17F have been described, as well in the IL-17 receptor A and an adaptor molecule, ACT1. And polymorphisms in the IL-23 receptor are some of the most commonly associated polymorphisms in inflammatory bowel disease, again providing evidence of the importance of this pathway in autoimmune diseases. There are many different therapeutics that are currently being brought to the market to target this pathway. There are therapeutics at target IL-12/IL-23, that is, p40, or just IL-23 alone, p19. And these are very effective in psoriasis and some forms of arthritis. Then, there are antibodies that target interleukin-17 alone, or a combination of IL-17A and IL-17F. And again, these have been very effective for psoriasis. And finally, there are antibodies that target IL-17 receptor A. Now, these are a little bit more complicated, because IL-17RA is shared by the IL-17A and F cytokines, with other cytokines, IL-17E… which is also called IL-25, which is made by… not only by type II… which acts on type II innate lymphoid cells that I’ll tell you about… and also IL-17C acts on the IL-17RE receptor, which is found primarily on epithelial cells. So, one needs a word of caution here, that we don’t yet know that much about the biology of some of these other cytokines and receptors. And this particular molecule targets all of these. ROR gamma t is also being targeted for therapeutic purposes, for the obvious reason that it’s upstream of these different cytokines, but that has not yet been tested clinically. So, ROR gamma t, if… when it is targeted, is going to affect not only the Th17 pathway, but also innate lymphoid cells. And I’m going to tell you just a little bit about these innate lymphoid cells that have been described only during the past decade. And we believe that these may have been early evolutionary precursors of the differentiated types of T helper cells. So, you can see here that there are innate lymphoid cells — ILC1, 2, and 3 — that mirror in their transcription factors those transcription factors found on the Th1, Th2, and Th17 cells. In addition, there are innate lymphoid cells that make both ROR gamma t and Tbet. And these are cells that have an NK, natural killer, cell surface marker, NKp46. And these seem to resemble very closely the pathogenic Th17 cells, the Th1* cells that I was mentioning. And these cells also express cytokines that are very similar to those expressed by the… by the T helper cells. And I won’t dwell on this, but keep in mind that the T helper cells and the innate lymphoid cells can share many different functions. I’ll show you one example here, for type II innate lymphoid cells, in which these are cells that have a very important function in protection from parasitic worms, or helminths. And what was found was that a very highly specialized cell in the intestinal epithelium, in the tuft cell, responds to some product of helminths and also of protozoa by producing interleukin-25 or IL-17E. And that acts on a receptor on type II innate lymphoid cells. And these cells will now make interleukin-13, which acts back on the intestinal stem cells, leading to production of more tuft cells as well as goblet cells, secretory goblet cells, which are very important for expulsion of the… of the parasites. In addition, the type II innate lymphoid cells have another receptor that selectively expressed on these cells. It’s a receptor for a neuropeptide, neuromedin-U. And that also is important in this type of regulation. An example of the expansion of these tuft cells is shown here, from work in Richard Locksley’s lab, in which they infected mice with a parasite, Nippostrongylus brasiliensis. They generated mice in which the red fluorescent protein was knocked into the IL-25 locus. And you can see, here, red fluorescence in rare tuft cells present, here, in uninfected mice, but a great expansion of the number of cells making RFP following infection. And this can also be shown by using a tuft cell-specific marker, showing expansion over here. But this expansion of tuft cells is probably not only dependent on their making IL-25, but there is also an effect on the type II innate lymphoid cells by neurons in the intestine, by the enteric nervous system. The enteric nervous system consists of many different cell types that are distributed throughout different plexi in the layers of the intestine, and they can communicate locally as well as extrinsically with the central nervous system through the vagus nerve. You can see here, with a pan-neuronal marker, that these neurons can extend into the villi in the small intestine. And it was found that there’s a class of neurons that respond to helminthic infection, through detection of patterns from these… from these organisms, through the innate immune responses. And they then produce the neuropeptide neuromedin-U. These are cholinergic neurons that also make acetylcholine, but neuromedin-U acts on this receptor on type II innate lymphoid cells, leading to production of interleukin-13. And again, interleukin-13 leads to expansion of goblet cells and tuft cells. And that leads, now, to more rapid expulsion of the parasitic worms, which are found in both the intestine and also in the lung. There’s another example, in type III innate lymphoid cells from work that Jhimmy Talbot in our laboratory has been doing. And what he noted was that neurons in the small intestine, here stained in red, are in very intimate contact with type III innate lymphoid cells found within structures called cryptopatches. These are sentinel posts just beneath the mucosal layer, beneath the epithelium in the intestine, and these respond to commensal microorganisms. And you can see that there are very close contacts made by neurons and these type III innate lymphoid cells, which are marked here with a knockin of the green fluorescent protein at the ROR gamma t locus. And what we now can appreciate is that these cells are specialized for making the neuropeptide vasoactive intestinal peptide, VIP. And what VIP does is to act on receptors that are selectively expressed on type III innate lymphoid cells, inhibiting their production of cytokines, particularly of interleukin-22. So that… we think that these are neurons that respond in a way to maintain homeostasis and prevent too much activation of this pathway, which can lead to hyperproliferation of the epithelium, and potentially can lead to damaging consequences. So, what I hope that I’ve shown you here is that the homeostasis at the mucosal barrier involves not only a couple of lymphoid cells, but also epithelial cells, various types of myeloid cells that make a variety of different cytokines, as well as enteric neurons, whose relationship to each of these different cell types is only now beginning to be elucidated. So, we are now in a very exciting period in which the various relationships between the different cell types can begin to be explored in much greater detail. So, I will stop there and acknowledge those in our laboratory who contributed to what I showed you here. Teruyuki Sano did much of the work on the Th17 cell induction in the… in the intestine. Jhimmy Talbot worked on the… on the VIP-positive neurons in the gut. And Ivaylo Ivanov, who is now at Columbia University, did the very important work, early on, showing the relationship between ROR gamma t and Th17 cells.