 All right, I want to thank Lida for inviting me and Phil for the introduction and I want to talk a little bit to you today about as you said microbial metabolites and as you can see from The structures and that's from Michael fishbacks lab There are treasure trove as we'll hear from Julian Davies tomorrow of both anabolic and catabolic Metabolites generated by bacteria and some of them have wondrous structures as you can see on that image And some are incredibly simple to carbon Carboxylic acids and I think we as immunologists are gonna see us in the next 18 months or so our recognition of how these microbial metabolites impact or affect immune cell function and yesterday Janet began to plant the seed I think as well as others about the importance of considering not only who's there in terms of taxa and structure of taxa and sequencing based technologies, but other omics based technologies such as proteomics and metabolomics for understanding our interactions as hosts with our microbiota So Peter mentioned notobiotics all sort of reinforce The concept of their importance with a slight twist. We saw important data from Sarkis Muslim onions lab today Previously from Ruth lay yesterday and Rob Knight mentioned it as well Notobiotics aren't only for the study of germ freemites, which are very important for under for us to understand how the microbiota affect the diversity of physiology in many different kinds of hosts, but they're very important to understand more complex consortia than just minimal microbial communities of one or two and These often need to be maintained these isolators and the mice within them across many many generations for us to understand the dynamic interactions and co-adaptation between an immune system or your physiological system of interest and The microbiota they're also not exclusive to mice So I don't want to give short shrift to other model system persons out there We heard from Andrew about the importance of looking at different Drosophila and there are flies and there are germ-free flies There are germ-free fish systems that are very useful and notobiotic fish systems and of course One of the classic systems for symbiosis is the Hawaiian bobtail squid and vibrio fish rise And I think all those model systems are really important for understanding basic principles of symbiosis and host microbiota Interaction that are important for understanding overall human health Okay, so more funding for notobiotic systems across many different Model systems and of course the repository as we've already sort of heard about the importance there of The other thing that I think is a gap the sort of maximal information extraction from some of these germ-free and notobiotic systems and I still sort of drive a lot of wonder and I think We can see from the recent work of Fred Backhead and others that even from germ-free mice that we've had for maybe 60 plus years There's a lot that we can learn so in the spirit of Ramoni Kahal We can with respect open up one of these animals postmortem And I think everyone can see just on the gross image the or the Big picture image not under the microscope But with our bare eyes that clear differences between a germ-free animal and a conventional animal and a lot of these differences Outside the gut haven't been adequately explored or tapped in mice and other model systems not something we can do in humans Personally on what I like to do is often take out organs of immunological interests Slice them stain them look at them at the flow cytometer and this Image is I should say from a wonderful review from Andrew McPherson And for many years immunologists have recognized that a variety of adaptive immune staff subcells That's such as CD4 positive T cells and CD8 positive T cells and B cells and plasma cells that elaborate IGA are markedly impacted by the microbiota But there are many other systems that still need further investigation and there are many many details that we are still far from understanding We've heard something about T cells From Dan Littman and from Sarkis-Mosmanian There are a couple of few things that I want to touch on One is that their behavior is very different based on where they are in the body that they have functional Specializations just like our microbes do so a strep pneumonia can act a certain way in our nasal passages But far different in our bloodstream and the same is true for all these subsets of CD4 positive lymphocytes There are specializations of T helper 17 cells in our small intestine versus our large intestine which Dan Littman Outlined yesterday. There are also a lot of spatial specializations of regulatory T cells The other thing that I'd like to highlight is that lymphocytes did not evolve to give us auto-immune disease or to influence our propensity to cancer But they evolved to help us live in this microbial world and also I'd say regulatory T cells helped us learn to live on different foodstuffs, which is offered up a lot of benefits from a evolutionary perspective so Enabling us lymphocytes are enabling us to live in our microbial world. I've become quite interested as many people have heard About today about regulatory T cells. In fact, I think there are at least eight talks that I heard make mention of them and a particular kind of regulatory T cell that I'm going to talk about today is the Fox P3 positive regulatory T cell and That word Fox P3 came up on a few slides So I just want to add a little clarity to what that term means. It's an important transcription factor or Master regulator gene that's important for T regulatory cell Development and function there are people that have alterations in that gene and they have Multi-organ inflammatory disorders that are quite serious and we have a variety of tools in mice Thanks to Alexander Rudensky and other immunologists that enable us to look with some ease at that subset in different animal models So we've heard from Dan Litman and Sarkis-Masmanian about different interactions with specific microbes and either T help or 17 subsets or regulatory T sets And if I would characterize a lot of these studies the multiple studies by Litman and Masmanian and by Kenya Honda's lab I'd say they've followed a particular line a very successful inquiry in terms of the information and insight they've generated But I'd been them into perhaps candidate microbe approaches so looking specifically with the intention of Identifying a microbe linked to a function So Dan went through in some detail how they learned about the importance of segmented filamentous bacteria for T helper 17 cell Numbers in the small intestine and we've known about segmented filamentous bacteria as many of you know Since the time of Dwayne Savage I think he first saw those under the microscope and using a phyletype array a kind of technology to discriminate the microbiota of Mice from different vendors. They were able to see differences in many clays between these two different mice and their feces but really one of the strong signals was in SFB and Prior biological knowledge because other investigators had worked on SFB Suggested that this might be a candidate microbe worth pursuing further Another example of that is Bacteroides fragilis, which you heard about from Sarkis, Masmanian So Bacteroides fragilis has been investigated for decades and really in the last decade Especially worked by Sarkis has established this really interesting zwitter ionic polysaccharide is a key symbiotic factor by which B. Frag influences the behavior and the multiplicity of regulatory T cells And that's been really neat to understand it to identify Not only a specific Molecule and organism that are important for the development of that subset But I'll be a little honest and that honesty is that not all people has have SFB I think many of us know that not all mice have SFB not all people have Bacteroides fragilis and They don't usually mice don't usually have Bacteroides fragilis other approaches that I'd put into the Candidate microbe approaches are the beautiful work by Kenya Honda's group So there was a lovely paper in science in 2011 where Kenya Honda manipulated the microbiota with Chloroform extraction to sort of alter the community to look at spore forming bacteria and perturbed the microbial communities with vancomycin to get at candidate microbes that might be important in shaping Regulatory T cell function and went on to identify in 2011 published in science a consortia 46 Clostridia strains that were important for regulatory T cell function and development and more recently in nature There's an e-publication from his lab That's narrowed that list of Clostridia to 17 and those 17 strains are all derived by people and Exert this function both in persons and mice in terms of affecting that regulatory T cell compartment Work in Andrew McPherson's lab. I think Made my group and me start to question other approaches beyond the candidate microbe approach for Thinking about what was influencing regulatory T cell function So one was that he observed which was important to me across many many different strains of mice that the germ-free State regardless of your genetic background of a mouse deeply affected your colonic regulatory T cell component The other piece that he identified was that if you have a minimal microbial community a community of eight They altered Shadler flora because even in the 1950s and 60s There was an appreciation for these issues of standardizations with mouse models that we've been talking about Standard procedures and the intention of the altered Shadler flora was to add uniform a data mouse model So that's a little background on the ASF. This is a minimal microbial community that can restore immunological function to a near-normal state in mice And this suggested to me that there might be prevalent factors common factors Factors may be not intrinsic to specific species like Bacteroides fragilis, but that existed in our guts that contributed to regulatory T cell function if you will and There is a fair amount of prior biological knowledge about what math metabolites are in germ-free mice and what metabolites are not in germ-free mice and While this is a very daunting figure. I think it's very important that we sort of Keep metabolism at the forefront of our brains when we think about how our microbes interact with us I was chatting with Peter before and I I remember I think I first saw this sort of very intimidating image of Metabolism when I was taking biology in high school, but I think it's incredibly important for us to be mindful of All these pathways going on in bacteria and there and our cons and the consequence for our immune function in general physiology so one group of metabolites Which we've heard a lot about our short chain fatty acids and there are roughly 85,000 papers or citations and PubMed that mention them and what we've recently found is that they're very important for regulating colonic regulatory T cell homeostasis and That's good for us because maybe I don't have B. Frag or I don't have all these 17 consortia of Clostridium in my gut But the vast majority of us do have functional and well-functioning regulatory T cells and by and large we're making short chain fatty acids So from whence to these come Whether we've had microbial physiology or not We've probably all heard about fermentation some many of us enjoy fermentation by yeast where we generate alcohol from simple sugars bacteria Assist us in the digestion of indigestible fibers and generate a lot of these short chain fatty acids Which include the two carbon acetic acid three carbon propionic acid and four carbon uteric acid and these have generally been linked to a lot of beneficial processes and Also have been linked to fiber consumption there's a vast and compelling literature about that and Decrease levels have been associated with the Western diet and I'm specifically referencing an interesting paper comparing the diet of some children in Burkina Faso by D. Filippo and PNAS and the diet of children in the U which examined both the microbial profiles of these two Populations and also their short chain fatty acid levels and there were correlations with Western diet correlating with lower levels of short chain fatty acids They're of course very important for epithelial homeostasis So the epithelium of the rectum particularly likes to use butyrate as a as a source of energy Okay, so we did a very simple experiment and an experiment that's been done before I think at least the first Reference for this in the published literature is from Tori mid-bids lab in the 1980s But in our systems and our germ-free mice and across multiple strains of germ-free mice that we have We wanted to observe if short chain fatty acids were indeed Reduced and the concentrations here are expressed as micro moles per gram Usually in humans the fecal concentrations of short chain fatty acids are in the 15 to 100 millimolar range We're just expressing them a little differently here But we saw that acetic acid propionic acid and uteric acid in our system in our facility Remarkedly reduced in germ-free mice versus our conventional mice. We've done this in Swiss Webster germ-free and SPF mice We've done this in B6 Germ-free and SPF mice and we've done this in Balb C germ-free and SPF mice and this was a start of Correlating this with observations about decreased regulatory T cell But correlation is not causation as we well know And so we wanted to delve into whether this was a missing link in regulatory T cell populations in germ-free mice So we did kind of a simple experiment. We have notobiotic germ-free mice of different genotypes We were very careful with their drinking water Which is sterile and we added short chain fatty acids to it ruffling in the concentration of a hundred and fifty millimolar in the drinking water We either added no short chain fatty acids. That's the minus sign P for propionate A for acetate B for butyrate or a mix And what we found is and then I'll show data in two forms One are flow cytometry dot plots that will show you the Population of frequency of a particular T cell subset this talk will Exclusively be focused on the regulatory T cells which express that important transcription factor called Fox B3 and CD4 and on the bottom I'm talking about absolute cell numbers So these are actual cell counts and so you can think of this as sort of relative abundance or population frequency like we talk About microbiome data very often or you can think of it as an absolute number And I think looking at these two metrics of different cellular populations are Very very important in immunological analyses or any kind of cellular-based analyses. These are from entire colon from basically distal to the secum to just proximal to the anus and on the lower panel each symbol represents data from an individual mouse and So talking about a gap and again extracting maximal information from technologies that already exist Flow cytometry, I think has optimally been exploited by immunologists as well as fluorescence activated cell sorting and They're a handful of microbiologists I'll give a shout-out to Peter that are really using Flow cytometry and fluorescence activated cell sorting and bringing it to microbiology in a really powerful way And I'd like to see more people of different disciplines Use flow cytometry because I think it's a very powerful tool to look at populations of cells be it bacterial or microbial or both So what we see here Now that I've sort of laid out a gap Which I'm supposed to do as per elitist instructions is that we see that propionate acetate butyrate and the mix Increased in the colon both the population frequency and the absolute number of regulatory T cells So that was pretty cool for us this was a very big day in the lab that we sort of put out a hypothesis did the experiment or the experiment ovax as Sarcust likes to say and we had sort of this direct association by adding a microbial metabolite and seeing a response in a cell population What about functional responses so beside population frequency and number we were interested in whether The short chain fatty acids did more and so a few sort of canonical ways to look at t regulatory cell function Are to look at levels of this master regulator transcription factor Fox p3 and to look at il-10 an important anti-inflammatory cytokine and so whether we took the cells directly out of the animal That had been treated with short chain fatty acids or we treated regulatory T cells In vitro we saw that both these important molecules from T regs increased So it seemed like short chain fatty acids and here I'm just showing you data for propionate increase both Fox p3 levels and il-10 production in germ-free mice The next thing that we wanted to do is we've talked a lot here about how antibiotics perturb the microbiota and Many immunologists have looked at how specific antibiotics and perturbing the microbiota affect immune cell subsets Kenya Honda had done experiments where he had shown that vancomycin markedly affected immune cell populations and we wanted to know if short chain fatty acids could rescue that observation so in these experiments we fed vancomycin for several weeks and Continued the vancomycin and then a few weeks into the experiment into receiving vancomycin We added the short chain fatty acids Appropriate the acetate the butyrate in the mix and we saw that addition of the short chain fatty acids was sufficient to rescue population frequency and T reg number and so then this pushed us in the direction of asking if this worked in germ-free mice and this worked in a perturbed Specified pathogen free mouse that we had perturbed with vancomycin. What would happen in our conventional mice? Would we augment? regulatory T cell populations and that's just what we saw so similar design we put short chain fatty acids into the drinking water We wait two to three weeks in these experiments all three weeks And we see what happens to population frequency and number those are the upper panels and we looked at a particular Marker of the T regs in these experiments IL-10 and we also saw that that IL-10 Producing subset was also augmented so wow you can add a microbial metabolite and Boost what's already there and again? We saw this increase in functional signatures as well looking at Fox P3 and IL-10 from T regs directly Isolated from the mice And here each symbol is going to represent data from many mice because you get roughly 20 to 40,000 T regs per mouse colon Or in vitro where I'm data represent Repeat experiments Okay, so not only do T regs sort of have Fox P3 is a functional marker and make IL-10 Their key job is to suppress other T regs So this is a classic assay that immunologists use you can label a naive CD4 positive T cell with a dye We could add in our T regs and add in our short chain fatty acids and we can track proliferation of those naive T cell subsets by looking at the dye dilution by flow cytometry again bacterial cell proliferation can be followed in a flow cytometer in a similar way and what we found Here Either looking at data from a single experiment or data from multiple experiments was that the short chain fatty acids Introduced into the media increased the suppressive activities of regulatory T cells now This could be by increased IL-10 production But what we think is going on is even though we add a fixed amount of T regs into the system in day zero One possible hypothesis is that the T regs are proliferating more So here we're actually labeling the T regulatory cells and adding or not adding short chain fatty acids And what it looks like is happening is that the short chain fatty acids increase T reg proliferation and In another twist of what these relatively deceptively simple short chain fatty acids do we also found that they up regulate An important homing molecule for a colonic T regulatory cells So the Litman lab has recently found that a G protein coupled receptor called gpr 15 is an important homing receptor for colonic regulatory T cells and short chain fatty acids seem to selectively boost this As opposed to other molecules that are important in regulatory T cell trafficking like ccr 9 alpha 4 beta 7 and ccr 7 that is not up there So it seems like short chain fatty acids have many effects on T regs IL-10 production of Fox p3 expression a selective chemokine or homing molecule as well as proliferation What is what is the receptor for this metabolite? I remember someone asking yesterday? Towards the end of the day What metabolites are important? How do we go about finding receptors? Well, there's lots of prior biological Knowledge out there that can be mined and in fact for short chain fatty acids There are a couple well-known receptors one is gpr 43 you're at far, too It is specific for short chain fatty acids as opposed to medium chain or long chain Lent fatty acids and it has a binding affinity for propionate that's greater than acetate or butyrate And that's why I've included on the screen mostly experiments with propionate the PC 50 is around three to four point nine Not fantastic as an agonist, but clearly effective on gpr 43 couples to both pertussis toxins sensitive and Insensitive G proteins and it's predominantly expressed on immune cells and there's been beautiful work from the McKay lab in Australia about its importance as a receptor for chemotaxis on both neutrophils and Macrophages, but if you look in the literature, basically everyone's does it's not expressed by T cells But sometimes you can't trust what's in the literature and sometimes it's good to look an engine and geo and a positive migra race And so we asked just that question. What about T cells? And so using quantitative PCR and flow cytometry We found that gpr 43 is expressed on colonic T regulatory cells, but not highly expressed on mesenteric splenic T regulatory cells and its expression is also dependent on the microbiota and the gf is for germ-free and the SPF is for specified pathogen free and that's what we're showing there What you're seeing here is we're asking the same kinds of questions about the phenotypes We observed with short-chain fatty acids and they're dependent upon gpr 43 So sometimes there's redundancy in important biological systems. So in the absence of gport gpr 43 You still have colonic t-rex, but you don't see that augmentation of t-rex population frequency and number when you delete gp 43 or have animals that are missing gpr 43 Also, we saw that expression of Fox p3 and production of IL-10 in response to short-chain fatty acid exposure Was dependent upon gpr 43 expression as was suppression and Then we wanted to get a little bit into mechanism because there is a tremendous amount known about short-chain fatty acid Putative mechanism and there's also a lot known about t regulatory Cell functions and what molecules regulate? Fox p3 expression what molecules play a role in regulating diverse processes in Regulatory T cell functions and a lot of those are histone deacetylases This is work of Wayne Hancock and Desitin and many others and specifically to histone deacetylases are really master Regulators of regulatory T cell function and what we found was that short-chain fatty acid in a gpr 43 Defendant fashion effect expression of hdak 6 and 9 which are well known to be important for Regulatory T cell function. We also found in a gpr 43 dependent fashion that histone Satellation is increased in response to short-chain fatty acids exposure so I think it's very useful often to think about Gastroenterologists and all their useful knowledge in the literature from their clinical experience and they have not only a vast knowledge and Experience with fecal transplantation dating back to at least the 50s but also with short-chain fatty acids and for many many decades short-chain fatty acid mixes have been given for a Condition called pouchitis, which we heard about from Eugene Chang and given for something called diversion colitis and in some patients They make better So we remembered that and we used a model of colitis that you heard about earlier today from Susan Erdman And you also saw some data from Sarkis-Masmanian and this model is useful because we can track short-chain fatty acid effects on T cells specifically in this model of colitis and what we found was the short-chain fatty acids when given to a mouse Protected them from T cell transfer colitis and augmented the effect of T regulatory cells that were transferred and Then we also established that these effects of short-chain fatty acids using T cells from gpr 43 Deficient mice or sufficient mice were really important for this effect In other words these effects of short-chain fatty acids appear to be both t-regg intrinsic and gpr 43 dependent So to summarize short-chain fatty acids are a class of abundant microbial metabolites that affect adaptive immune function specifically t-regg numbers and function and appear to ameliorate intestinal inflammation So challenges briefly got microbial metabolites Which ones affect my mucosal immune system responses? The NCI has an RFA about discovery around studying microbial metabolites in GI GI malignancies and I think for whatever your system of interest is you could sort of fill in the blank That's filled here by mucosal immune system development and function. What are the host cell types and receptors for these microbial products? I've given you one example about how prior biological knowledge can get you faster to answer in these questions And I think a corollary to all this or what host processes do our microbiota listen in on and I think we heard examples in terms of sex Hormones in terms of estrogen and progesterone Reactive oxygen species are another example of that and there are many many examples of different molecules that we as hosts make That might affect the microbiota So with that I'd like to acknowledge the members of my lab particularly Pat Smith whose work I've highlighted today key collaborators and of course the NIH for funding not only for funding research in the lab, but supporting students through T32 awards and Postdocs through T32 and F32 awards. Thanks Thank you very much Wendy, and thank you for the plug for the NCI PAs I think the data on short chain fatty acids is a very nice segue into our next session Which is on the diet and its effects on microbiome Do we have any questions from the audience very quickly a great talk just one quick question What's your idea for a biological model? Why would the immune system depend on on? Metabolic compound to regulate such a fundamental thing Why would the immune system? No, I mean Why are the why specifically short short chain fatty acids? Why are they so so important do you have any idea why that could be? I think they're an abundant metabolite, and I think when you're thinking about how a molecule has to work at a distance Concentration can be limiting. I think many microbes make short chain fatty acids and that explains why if you were going to be dependent upon in some way Metabolite you'd want many microbes to potentially make that I don't know I answered your question, but I think it could be fun to further discuss during the break One more question, please Excellent talk just two questions, which are actually related So do you think that your short chain fatty acids actually reached colon and the other question? Have you looked at t-rex in the small intestine especially in Jejunum? We think they do reach the colon and I want to sort of call back a comment yesterday by Larry for me that I think It's very important to think about metabolic flux, and that's a key gap We also measure short chain fatty acids throughout the small intestine by looking at intestinal contents, and they tend to be a 10th or less, and we don't see effects in the small intestine In these experiments, and we've done the same thing for the small intestinal regulatory T cells as well gpr43 expression is about a Half to a fifth lower and the short chain fatty acids are lower. There's also faster transit time Through the small intestine as opposed to the colon of the intestinal contents Oh, hi, just one one one question a nutritional based question Very elegant work and fascinating, but I'm thinking about the way you introduce butyrate into your animals You put it in the drinking water I believe at pretty massive levels, which means you're exposing the mouse Systemically to butyrate which is not a normal or physiological state For example people who have delivered fiber for the process of fermentation in the gut Haven't seen these kinds of changes in t-rex cells in the colon And I'm just wondering whether you're seeing really a systemic effect And you already indicated you have a homing receptor that could be responsive to it And maybe that's what you're driving to the colon a massive recruitment of t-rex cells Which you don't normally see when you introduce fermentable fibers and things that might increase the luminal levels of butyrate What do you think of that? So we've also given the short chain fatty acids Intrarectively and seen the same results and we have some ongoing responses with fiber I would agree that it perhaps is an optimal to put this concentration of acetic acid propionic acid or butyric acid into the water if we'd want to Realize this for other treatments because it's not that palatable Although I'd say that this is about an eighth of the concentration of vinegar if you've had a table vinegar We have looked in the thymus and I can talk more specifically with you about the T regulatory cells because we've examined other markers that make us think that perhaps it's not promoting So much homing to the intestine, but it's causing proliferation of the regulatory T cells that are already there and a lot of those experiments come from looking at the marker Helios Okay, thank you for the excellent discussion excellent questions Bidna And I would like to thank all of the afternoon speakers. We will reconvene here at 3 45