[Music] so first of all thank you very much for inviting me to this meeting here as you've heard I'm not a nutritionist so I don't understand much about nutrition we focus on the immune system of the chicken only and what I would like to present is just one single aspect of what we do in our lab which is related to the development of the immune system and if we look at development that really starts immune system development really starts in the embryo it follows precisely genetic program people had tried to influence that but there is not much chance to really do that but then the birds hatch and we know there's still a genetic program going on with regard to the development of the immune system but there are also a lot of environmental factors coming into that so breeding companies are really interested in that part while I think nutrition companies are interested in that part which is environmental impact in general and nutrient impact on the immune system in particular the question is what are those cues and what we've done is we've looked at in particular the cecum and the cecal Tunsil this unique lymph fluid structure in the chicken gut which are two main target structures to look at immune system development and impact on this development and as we have heard today and I don't have to allude too much to it that the this whole gastric system is very complex it does not only exist of the single barrier layer which we heard it also has nerve tissue which is interacting with with the entire system very closely and I must say we know very little about it in the chicken system and even in mammals it's it's just something that is now much more recognized and we have the immune system and the immune system here it's as we heard before today it's a largest immunological organ in in the entire body is in the Duster intestinal tract and as you can see here in the lamina propria of a chicken gut you see all these brown dots these are only macrophages it's just one sub population of immune cells and you can see how abundant already this single sub population of immune cells is in this gut tissue if you look closer and I got this picture from colleagues in Bristol that's from swine and you see three color immunofluorescence staining and you see lymphocytes and macrophages and B and T lymphocytes and macrophages so it looks as if the entire villus is basically filled by the immune system which is not true we also have vessels we also have nerves and all the other blood vessels but it's pretty packed so I'm as an immunologist I'm always fascinated by the fact that we have a single layer barrier if you think about other barriers like the skin it's multi-layered keratinized barrier very thick but in the gut and in the lung it's just a single barrier we heard about the mucus on top of it and what we are focusing on are the many many many different types of immune cells and just to name them very quickly we have macrophages sitting here we have the dendritic cells we have T lymphocytes B lymphocytes we do have natural killer cells we have all kinds of granulocytes in there and from the viewpoint of an immunologist it's not just the T cell there are a lot of different subpopulations of T lymphocytes called T helper one cells tear but two cells here 417 cells T regulatory cells and most of these cells have been described they all have been described in mice we have some indication that they are present in the chicken but for many of these cell types we have not yet found the markers to really address the functional properties so that is in particular true for regulatory t-cells which play a very important role in maintaining homeostasis in the gut so regulatory t-cells for example are very important in in dampening down over reactions towards the microbiota which we have here because most of that most of those bacteria in there I would call the good ones and we don't want to have those getting into this area here and inducing an inflammatory response so some of these cells in particular the dendritic cells have a very unique feature at least in mice and we think they have the feature also in chickens because they have those extinctions extensions and what they can do with the extension so they can go through the interim period space and reach out and grab those bacteria and pull them back and the question is why should they do all the very simple answer is to present those to T and B lymphocytes but in such a way that the response coming out of that is not an inflammatory response but an anti-inflammatory response to dampen it down so to maintain homeostasis preventing inflammation that would be the first and most important function of this entire mucosal immune system it's only when a real pathogen makes it across the barrier and if we talk about pathogens we will hear later on more about Cydia for example we've done studies with Salmonella that's beautiful within days within one day you have an accumulation of these cells so myriads of these cells go into the gut and fight against that pathogen which was able to cross the barrier but most of them never ever cross the barrier only if they were pulled into that space on purpose by the immune system so we've heard about the importance of microbiota and fermentation we know that nutrients are taken up here what is interesting that nutrients also have a major impact on the epithelium we know that butyrate is for example a very important energy source but nutrients are also impacting on the immune system we may humor about it later on and then also the microbiota is impacting on the immune system and they say most of that work like 99.999% of that work has been done in mice very little tiny bit in swine and we try to do some work in the chicken and what we should keep in mind that these cells in the immune system are also communicating with each other here in this environment and they are also communicating with epithelium and the epithelium itself is again communicating with the cells of the immune system it's a very complex system and it's it's rather difficult even for most people to understand that and to get a hand on this very complex network of interactions and then what do we do we have all these nice ingredients here antibiotics probiotics prebiotics phytonutrients immunomodulators and we dump all that into the gastrointestinal tract and the immune system has somehow to cope with that and we have very little understanding of what happens here really so what we did was because it's way too complex for me to understand we thought we should break it down to a very simple set of experiments which already had been done over and over again in mice but I should say one of the first experiments has been done in the chicken system way back in 1956 by Jeannette thaw back in New York and she at that time found cells which she called T suppressor cells and these suppressor cells are now our famous T regulatory cells nowadays so what she and what we did was to raise Birds basically without microbiota we heard about germ-free Birds earlier in Isolators and that was only possible because we had that fantastic collaboration with the people at in rot or did you have these birds and the Isolators and we compared those with SPF birds to see how the immune system develops under the impact of the immune system we did all kinds of essays I break it down I don't show you everything but what we did first we did micro arrays at that time and we wanted to see what is the difference in gene expression patterns between germ-free Birds and SPF birds very straightforward approach what was interesting and I don't go into that further but was interesting these birds in comparison to those birds expressed a hell lot of solute carriers so they were all up regulated all kinds of solute carriers and since I'm not a nutritionist they don't tell me much but if you're interested I give you the data and you can go ahead we have all that as immunologist we were obviously interested in the pathways called immune function and developmental processes and what we found right away was that if we compared germ-free and SPF Birds we found very strong regulation of genes of the immunoglobulin family we found a lot of factors up regulated which play a very prominent role in B cell function and development and I'll give you an example this here is an anti a pop-top tech factor for b-cells it provides survival of b-cells in that environment we found a ID most of you might not have heard about that enzyme but that is the most critical enzyme in order to get antibody diversification to make sure that we can respond with good antibodies to almost every antigen without this we will die no chance to respond to foreign antigens and a number of chemokines and chemokine receptors and come back to this later on so obviously the first thing we realized was that the microbial colonization of the gut very much in acted on the B cell system and antibody production and so rather than focusing of that on the cells of the innate immune system we really looked at cells of the adaptive immune system and at the B cells in this context initially so this gives you an example of germ-free Birds with SPF birds and that is standing in the gut for macrophages and as you can see easily there is no big difference okay immunity is not quantitative but as you can show you other pictures which are quantitative so not much of a difference so it looks as if this part at least of the innate immune system is following following the genetic program regardless whether we have bacteria or not if you look at the B cell system as I said before huge difference look at the SPF birds at 3 weeks of age we see this accumulation of B lymphocytes and later on the formation of these organized structures which are called germinal centers these are the structures were the good piece of responses are initiated during vaccination to make a good antibody none of that happens in germ-free birds they essentially let B cells in the gut you see some brown dots that is only due to the fact that our antibody is not only staining B cells but it's also staining a minor subpopulation sitting in the epithelium which we have no idea what it is but it's not B cells so what about production of antibodies which is the primary feature of B cell function and as you can see here they don't produce IgA at all well IgA most of you might be familiar with that is the number 1 quantitatively the number one immunoglobulin produced in the body we find very little in the circulation in plasma but we find a lot in the gut it's gram wise secreted into the lumen of the gut so quantitatively IgA is the most abundant immunoglobulin produced by B lymphocytes and as you can see here germ-free birds cannot produce IgA well that's really not that much of a surprise if they don't have B cells that can't produce IgA so obviously microbial colonization has a very significant impact on B cell development and it would be interesting to understand which species are actually driving that so what we thought is we take germ-free Birds and dump in a couple of bacteria so we added another group to this and that was monoclonal in ization by just eco lion and we picked a particularly coli which is sold in Germany as a probiotic in each and every pharmacy as a human being you can buy it and eat it and it's really famous because the story behind it is that actually is called ecoli nestle 1917 because a a German doctor at the military camp which came down with diarrhea everybody came down with diarrhea found one single person without diarrhea and from that person he isolated that a coli so it has been around since 1917 and that has now been studied because of its unique properties very intensively so we thought we pick that one because there was also evidence and the most literature that it would be interesting that again our simple analysis you can see here macrophages again germ-free the single E coli conventional not much difference I would like to point out one thing here we don't see inflammation so we have a bird without any bacteria we put in a bacteria and the immune system is maintaining homeostasis and not responding with infiltration of inflammatory cells it's not responding with a classical phenotype of inflammation which would be blood vessel enlargement leakage of blood vessels and so on and so on it's just maintaining homeostasis and if you compare with the conventionals okay that all looks the same what about B lymphocytes or T lymphocytes in this particular case well they don't have many T lymphocytes here while the conventionals have nice aggregates of T lymphocytes so the whole system is also not only affecting the B lymphocytes but also the T lymphocytes what about B cells no response so we thought mmm maybe Ecola is not sufficient what about for bacteria and that is pure guessing I mean we had no evidence we had no no real reason to pick one or the other except for e.coli lactobacillus Enterococcus and Clostridium there were all these strains or all probiotics licensed probiotics so we've thought we dump them into the birds and see what happens and the first question was do they colonize and as you can see here they all nicely colonized so we got pretty high numbers in the cecum after colonization in those birds so that worked the question again what was the effect on the immune system not much on the macrophages here again as one bacterium here are the four bacteria they seem to do something so it's improving for the b-cell system here improving not yet there but improving if you look at this particular organ the the sickle tonsil which is an organized lymphoid structure in the chicken gut you see how small they are in germ-free birds and comparison with SPF Birds here I call them conventional these are SPF Birds we do see some impact of a single Ecola here in this particular organized lymphoid structure because they start to develop very small and very few but distinct germinal centers it's even better with four bacteria it's not yet as good as in the SPF birds so there's still room for improvement and the same is true for IgA and we thought we should quantify it and the easy way for immunologist is to measure antibody levels so what we did we compared i GM plasma levels in germ-free birds and in birds with four tetra reconstituted Birds and the SPF Birds since we did that in Isolators we had only small numbers of birds only five and you can see here there is a tendency towards much better i GM production also towards much better i gy which is the equivalent of IgG and the chicken and so it is for plasma IgA so that is plasma what about IgA concentrations and the seeker content the plasma goes up but also the content secrete that the IgA secreted in the mucus and we heard today that IgA seems to be quite closely connected to the mucus and actually captured in the mucus and also in the further into the gut content we can measure that and find that they nicely respond so then we thought do birds which get just the single ecole might do they produce antibodies specific of e.coli I told you they don't show any inflammatory response so the question was do they respond functionally at all and what was interesting if we compare the germ freeze with those who get the e.coli only they made very nice anti ecoli antibodies which were secreted into the gut lumen so obviously Monica Lola's ation did not lead to a full-blown development of the immune system but it really activated the b-cells system to produce ecoli specific antibodies and if we did the Tetra colonization you can see here titles were much lower which makes in a way sense because in this case e coli was just one of four bacteria and maybe the immune system was also taking care of the other ones and the SPF birds were very low but that it might be due to the fact I'd show you later where that is we looked at I do I pretty much the same pattern and we looked also against the second bacterium the Enterococcus faecium which was added to the tetra flora but you can see here the response to that particular one was not really all that good so this e coli seems to have unique properties to drive immune system development in comparison to the Enterococcus and the same for Enterococcus is true for the igy in the chicken system well that is thought to keep it as as easy as possible however some some more gene expression studies related to the B cell system you can see here we could confirm but qPCR that the iga transcripts are driven up and if you compare SPF to mono reconstitute that then tetra reconstituted it's really driven up it's much more control than the SPF birds and that is also true for the iqy and we looked at some of these factors we found in our microarrays to confirm what we've seen in the micro arrays with regard to this b-cell NTA popped otic factor and this is fold induction in comparison with germ freeze so birds without any bacteria in comparison with SPF mono reconstituted tetra reconstituted so this important piece effector goes up so does another important regular tour regulator of t-cell to cytokine called cd40 ligand also a very important piece of cytokine called il 21 again this important side of the enzyme which is driving diversification of the immunoglobulins interleukin 6 goes really up as you can see so for most of you in the endo trishing business the simple answer here be Oh aisle 6 goes up that is inflammation well aisle 6 is kind of a very complicated cytokine because it's really playa tropic and has many many different functions and one of the most important functions of l6 is actually to regulate the immune system rather than just being inflammatory il-1 beta TNF alpha they are much more inflammatory and much more restricted to the inflammatory pathway in comparison with il-6 which is a very important cytokine for example for b-cell development and also for the development of regulatory t-cells and then we have aisle one beta and if you look at fold in Dutch in aisle one is induced but not very much in comparison to aisle six which is 50 fold up and that is only five fold up so there is no strong inflammatory response going on so that is further evidence that this induction of aisle six is much more immune or regulatory rather than inflammatory so when we arrived at that point and we have tons of data on that but when we arrived at that point we thought well this is what we had been looking at up to this point this is what mother nature does so they both hatch but then the environment is entirely different and we thought we should try to to run an experiment where we give young birds a chance from hatch to pick up a maternal flora saying that in mammals it's it's natural that they will get a maternal flora from the beginning so every piglet every human being will be in contact with the maternal flora right during birth process actually already what we do in the chicken world is entirely different we keep them as clean as possible as long as possible and then well not in the u.s. though I know but this is not what we see usually so in establish maternal flora so we thought rather to compare germ-free with SPF conditions with what I now call Convention on natural conditions and now looking just at the IGA concentration of all the histology and everything but everything shows what is shown here I told you we had a very interesting difference between SPF and germ-free it's very low they should compared with the conventional birds so a good maternal flora is really driving the development of the immune system even further and that is not only true for IgA and plasma but also for GA and the Seco content and because these were again only five Birds and we never we never ever immunologist usually work with three to five animals not like nutritionists but we thought we'd do a real sensitive immunologist study not five thousand but let's say 20 and we repeated that experiment and as you can see here the SPF Birds produced IgA but those with the maternal floor um were much much better so we went up for 500 units all the way up to 2,000 units so threefold induction which is quite a bit with regard to quantitative induction of immunoglobulin the other interesting point is we immunize both those here and we immunize the group here an immunization did not drive up the total immunoglobulin level it drove up the specific immunoglobulin level but not the total immunoglobulin level so the impact on total immunoglobulin levels brought about by the maternal flora is much much stronger than any other stimulus we can come up with I think and that is also true for the cecum and just what is the role of IgA where I'm talking today to today about IgA basically IgA is secreted in large amounts it's transported it's picked up by the Pagliacci receptor transported across the epithelium and it has basically two functions in the gut first of all for a long long time IgA was considered to be important to control pathogens so it can bind to pathogens and avoid pathogens from crossing that barrier and then there will be flushed out and that is one of the very important functions if they're true well there's a little problem with that and the problem is they're 1 in 700 humans have a selective iga deficiency they don't make IgA but they don't die and they have very very few infectious diseases however what we learned later on was this receptor can also transport IgM across that mint barrier and in those with an iga deficiency IgM is taking over I recently talked to a guy in Aachen who is one of the leading mucosal immunologists and he told me that he is now looking into infants submitted to this university hospital with severe infections and in this case one in 30 of these kids has a selective iga deficiency so it seems to play a very important role in controlling pathogens in the gut but there is a second important function and the second important function is to bind to bacteria in the gut the commensal good bacteria in the gut why would that be important well it's controlling the composition of the microbiome making sure that the system is not screw towards one bacterium but the entire micro flora in its complex composition is maintained how do we know or we don't know in chickens but what people have done in mice is they generated mice which have no idea then they again generated those mice to produce a monoclonal antibody iga antibody against one of the bacteria in the gut and then they could show that was entirely eliminated so IgA is very important in controlling the composition of the commensal microbiome in the chicken so what I've shown you is that this is a very important function of the microbiome in the context of the immune system it also impacts on the T lymphocytes I have not shown so the basic question was what is the difference between the maternal flora and the SPF flora from in rat or and here the data sequencing the microbiome profiling it this is the SPF group and that is the group of the maternal flora so you can see it's it's really different so what is the difference well I skip this and you can see here the major difference is we found only from acute is here in the SPF flora a lot of an aerobics but we don't find all the other guys very few of the Proteobacteria and none of the bacterial eaters and actinobacteria so this SPF Flora's really screwed so what we can learn from that because the SPF floor was not entirely sufficient with regard to driving development of the immune system this flora here and the bacteria sitting in here would not be a choice for good start of flora for chickens so the good start of flora would be sitting somewhere in here so we have to break it down further and first of all we did richness analysis and you can see here the richness as much worse in the SPF floor in comparison with the maternal floor out you would expect that we've done that for a number of birds and what we are currently doing and that is our approach to the whole system is we have isolated now in collaboration with microbiologists roughly 70 different bacteria from the gut they are all cultured and they are characterized and we found five or six really new ones never described so pretty much chicken specific this strain collection will be combined in a fermenter to give a give a cocktail of five or ten and then we want to dump it into chickens and see what it does to the immune system to the development of the immune system well and with this I finish we want to go from here to there but with a defined flora and I would like to thank my collaborators in particular the people that in rhetoric at the shul and Philip felt you you really helped me a lot with with a germ-free experiments my colleagues in arc and who are doing the microbiome stuff and now generating those cocktails which we can then dumped back into the birds and then my people in particular the PhD students are letting on on that project who did the actual work and I have the privilege to talk about it thank you very much thank thank you sorry to disturb you just one quick question just pick one of them yeah very good question we obviously we looked at that we looked at the ileum we looked at the colum we looked at the jejunum we did not look at the duodenum but the the composition we did microarray studies on the jejunum jejunum and ileum and there was only a minor response to that so the cecum definitely is I think the place to look at what we don't know yet is where is all the education of the immune system going on and what we don't know yet is what are the signals to get the immune cells in particular the B and T cells into the gut so there must be signals from the gut to the blood to tell the blood lymphocytes to home into the gut and to stay there and to do their job and we have no idea how that works and whether educated to do that but it's clearly the secret to look at [Music]