[Music] and I would like to introduce dr. febray Sloane is the immunologist and has been working with in residence 1994 and his focus is host-pathogen interactions and intestinal immunity the floor is yours thank you for the introduction Thank You P Andre for this marvelous meeting and for this invitation in my laboratory we are actually interested in hospital gene interaction and we study mucosal immune responses and basically we do work with to infectious model one is affecting the livestock which is cryptosporidiosis and yeah I wanted I think the poultry industry that is coccidiosis we heard about already this morning what I would like to discuss with you is like yeah I would like to say that it's very important to investigate this immune cells immune effectors together we have the micro flora that they all together impact the outcome of infection leading to protection or to a visual pathology so I will use some example for work we have done this last year in the lab to show you how we can illustrate this but before I go with this infectious model just want to state that a tomato status is adverse when we we have the first incoming antigens coming from the floor from the flora of course but also from the food and this gave the very first signals to epithelial cells in the residing in immune cells and these what we can call the physiological inflammation we have some answer production of mucus of antimicrobial peptides and some IgA but this is thai quickly tightly regulated and there is no influence um kind of information but when we get an infection some kind of enteric infection of course we get an inflammatory response from this video sales but also from the immune cells that are coming into the mucosa and if it if it works as you expected you had a quick protection and then you go back as soon as possible to me or studies but if you have unregulated immune response then you get some pathological inflammation and you got a pathology with intestinal lesions okay I will jump now on the first model that is Cryptosporidium in this as you can see over here you have this extra still on stage of apartheid that will jump from AP ourselves to a PT or sense you to replicate if you worried about Cryptosporidium because I know many of main people here are interested in food food maybe you don't know the parasite so it's a single-celled parasite that just scroll on top of epi do cells and make its whole cycle in three to four days it's really restricted to the apex of epithelial cells and there's a high prevalence in the livestock and it's also a zoonotic disease it affects young cows and young children and I'm so inefficient amiduos because the severity of the infection is really linked to the immune status of the animals so it's a beautiful model to study intestinal immunity especially in neonates and these acids we tried to decipher all these mechanisms that involved in protection off is your pathology just just to show you how look like intestinal villi this is a normal one and did you see all the parasite all along and the actually all epithelial cells can be infected so of course this dramatic change in the regulation of these cells so what happened when the first deposit first and couch of the epithelial cells if your cells just give a first answer they just produce small amount of antimony peptides and they can undergo Buddhists there and using their own cell deaths but usually this is not enough to kill the parasite especially because the plant has developed evasive mechanisms to most of these even mechanisms of killing so but I also a pity on cells they produce a small molecules that are called chemokines and these chemicals attract immune cells in this case this human cells will control the infection but we we try this noise here to understand which of these cells were important in the protection that's why all that we used a mouse model because we can use a lot of different transgenic or knockout mice to try to decipher the complete mechanism the first set of exponent that we did was to look at use mice that are deficient for T or B cells in this case you know this is a curve of oil you see the excretion of apartheid there's a acute phase of the infection and then the animals are protected and when you don't have this B or T cells you also see the exact same curve actually he adaptive immunity is only important to completely all the infection but it's not important to control this acute phase of infection that is leading to this diarrhea in these young animals so we knew from that that was innate immunity was key but which cells and we first start to look at molecular phagocytes as it's a large family that you can find some macrophages or dendritic cells and we wanted to know which were important if these cells were important in which one so the first model that we used was you some CD and CD two mice whatever name is for you just remember that with that model we can have a transient depletion of this all mononuclear phagocyte all this family dendritic cells monocytes and macrophages they are all depleted sometimes almost 100% for dendritic cells and for this one almost 60% of our sales on depleted and with this model you can clearly see that the curves after depletion you got a higher level of posit envious Tenten be intestine of his mice obviously no late mouse but then as it's just a transient depletion we've added shock seen the new cells coming from the bone marrow just come back and then you regenerate all these subsets and then the neonatal mice can control the infection so for sure now we knew that at least dendritic cells or monocytes and macrophages we are clearly important through control these acute phase of infection and we continue to work with these different models and to differentiate if there was a macrophage or dendritic self we first focus on dendritic cells and we use different cytokines where you can amplify some subsets for example and you inoculate fact-free ligand administration so you just amplify this different subset by doing this we were increasing the resistance to infections so what's a good fantastic that these cells were very important and very nice V which is published paper months ago as I mean and we which for that we use battery deficient mice these mines have deficiency for the development of some of these subsets of dendritic cells and a clearly this city one of free dendritic cells are really at present at very very low level and these mice clearly are much more infected at the conventional one and if you just even look at this young animals five weeks later you can still see the partite in the intestinal villi so with all these between models now we knew that this dendritic cells were really key for controlling the infection the mechanic by which they do that it's probably because they produce not amount of il-12 and actually the first and you won't get my response that it's very important cytokines to control the parasite in the epithelial cells and probably this signal will be amplified by over innate cells like in 18 for site but at the same time we we have found that there's all so a lot of inflammatory monocyte that were recruited at the site of the infection and to study them function we we just look at we use some another model that is deficient for CCR - it's a chemical receptor and in this case in biz mice the monocyte cannot get out of a bone marrow and they cannot be recruited in the lamina propria so there was a good model to study that function and with this mice that only very very few inflammatory monocytes although these mice monocytes are present in very high level in normal mice and they're in close contact to infected epithelial cells we have this model we were expecting that the mice will be much more infected like we have seen previously with the vision of dendritic cells and it was not the case at all so it was a bit surprising for us we see how this 13 fold increase in an inflammatory monocyte do nothing there was a bit surprising so we with some thinking maybe they're not involved in protection but maybe they involved in some deleterious effect on the mucosa so as we know that Cryptosporidium is inducing diarrhea and we know that they're also verifying the transition resistance we we have done experiment and in vitro it's clear you can see with this trial chambers you infect with crypto and you see a drop in the transitional resistance we have done this also in vivo well we'd use a different mechanism we use fitzy Textron and then normally at most as is there's only few of the fits addiction that you give by gavage that can go through the if your cell thing go through the blood and you can measure the fluorescence in the blood but when you do that with infection with crypto you clearly see an increase and it's depending of a variance of strain of crypto you are using so knowing how this we also made some experiment with a postdoc and he was able to make a long story short that Cryptosporidium by itself on epithelial cells was so affecting some of it at events Junction and in case eeeek Iranian beta-catenin and in vivo we found that this mechanism is probably due to the very large amount of Iowan alpha and gnf alpha that I won't be tnf-alpha that is produced by this inflammatory monocytes and as I was mentioned this morning these two cytokines are able to destroy the tie Junction and then for explaining why we have this increase in permeability so to imagine the very first part so just imagine for this model you're not Cryptosporidium coming into a pity your cells and the chemo can subtract these immune cells invade I mean appropriate some of these cells are more involved inflammatory monocytes and more involved in these streets of permeability and we know that this acute diarrhea in young cow so maybe this one of the mechanism and some other source of my chains are actually protecting the epithelium likely stiffen the subset of dendritic cells by producing in different gamma and there's a lot of different interested on mechanism that can block the deposit development in response to mass of gamma if you want to have some review we made that review last year so far to have want to have some more information please go and read it ok so what how can you use this now this information to see how we can strengthen this mu cosine in neonates so we knew for my probably everyone's know now if the audience now what are the so like receptor so innate receptor that are present on the surface of a cell or on those small compartment and and when you bind some other different ligands coming from bacteria or viruses parasite infamy then you induce some inflammatory signals in the cells and a chance now they have also new synthetic ligands that are available commercially it so you can buy and try this so we have to own its quite a long different experiment with different toll-like receptor agonist and we have shown that when we just inoculate the Saigonese to the newborn mice we were quickly protecting from against Cryptosporidium and this mechanism sometimes you can have a 90% protection in less than 24 hours so it was very efficient but when I wanted to share with you is like this with different models we got different type of freshman's what was interesting is like for CPG or the end like mimic bacterial DNA for exam we got a protection and for the IC that mimic some nuclei seed from viruses you got also a protection when when you do an antibiotic treatment with a broad spectrum of antibiotics removing almost all bacteria then you lots of protection with that with Puli I see and you don't lose it with CPG you at the end and it took a while for us to understand how this was working but in the case of Foley I see that some signals coming from fragile a bacteria that binds to Tallaght receptor v that is present on the surface of ister dendritic cells and then induce some mi di d8 signaling so a different set of cytokine expression and that together we have a first signal give protection but if you remove that signal this pathway only is not protective so and this is not the case for CPG because this goes and for strong activation of MIT ADH pathway so now a force base versus just a proof of concept but we are just seeking for a natural compound that and just make the same effect I will now jump on the second model so coccidiosis and it's just a picture of sexual stages flagellate stage it the male they will try to find a female to make an oasis sorry okay we heard about coccidiosis this morning but just to say a few word about the disease it's ranked it in the top 20 vet disease and it's most prevalent this is affecting the broiler industry there's a strong and economic impact it's a three billion dollars per year and due to production losses and treatments we do have a vaccine for layers but it's bit costly and cos T and we we have Kemet repeal so for progress that is mandatory because the the disease is very prevalent but there's a lot of resistant to all these molecules so we're clicking a sign of coccidiosis is decrease groups weight weight losses mortality and decrease at production but today I would like to focus on one of a strain that in fact one particular place at the intestinal tract of chicken is like a Marietta nila that close emerging sickle coccidiosis in young poultry and we wanted to understand what was making we would like to understand what was the mechanism for this a matriculate imager mahadji cyclic lesions for that we have developed several transgenic my lines for Ameria that we have expressed fluorescent and we now do have also unique to for screaming of and he may react compounds in the lab so the working of i proteases was like a normal condition then Ameria infected video cells start to develop some of a science go back in the lamina propria and then their release some free stages and probably we expect some strong inflammation because it's within the lamina propria probably inflammatory monocyte maybe like we have seen with a previous podium model and this will increased intestinal damage and may be translocation of bacteria from the commercial flora will increase this information leading to this huge lesions that we can see with infections model so we first look at macrophages and you can see here we have between apartheid we are being silenced you got macrophages that comes all around these sirens so we do know that these cells are recruited to ring the infection around the infected sites and also that these macrophages when you isolate them they produce I knows which is an enzyme that will produce an O and superoxides so in these cases this so these agents can call lesions but we still have to demonstrate this so you see that the importance of microflora we use an axe enoch chicken we're actually producing roast chicken so broilers without any flora and then we infect it with ma Atena and look at the lesions that was very interesting to see that compared to normal animals the actioning one have almost no lesion and even if you increase the numbers of the oasis at an inoculum ten times you see there's no lesion on the signal cooks to you so that was very exciting for us and we say maybe the the bacteria fry is making it all but the things are probably much more complex because if you look at the numbers of apartheid that replicate in these actioning animal it's much lower too so the next challenge for us now to understand what is the mechanism we should determine what is due to parasite replication for the lesions and why it is going like this or it is due to the bacterial flora that in maybe some breaches in the mucosa in the mucosa and some bacteria can come in Simon labrum propria or maybe both so what we do also it's just an ongoing project of this goal integrity project we do work we've also from our colleagues that also work in tool but nutrition is I am not so I'm not going to go into much details but what they want to do is to modify the element ation of young birds with insoluble fibers in the starter feed for the first days and we will see it by changing the micro flora we do impact the lesion score at the end something I would like to share with you also is that do good and bad guys exist in immunology and bye guys I mean immune cells or effectors and you will see that we actually all cells can be good and bad at some times depending of the condition I will just illustrate with this with another infection by photos and highlight that it's Toxoplasma gondii i and if you look at the literature you can see that for example if you infect orally mice see 57 miles which Toxoplasma gondii i you can produce a huge entire gamma production by cd4 t-cells and this huge in for example production in the nominee appropriate will induce highly itis and mouse death so if you just look at this data you will just conclude that interferon gamma appears deleterious to confer controlling the disease but if you go back and write a new see how many papers are published showing that for example with attenuate strain of Toxoplasma gondii i if you use mines that do not produce interferon gamma they will die quickly and if you look at how the mechanism is usually all this anchor from gamma produced by diffuse cell immune cells they will probably they control the partite into especially into macrophages but in different cells so for these you can see that every molecule depending of the condition could be beneficial or detrimental for an immune responses so these depends of the part of the ambulance the host genetic but also different what I call in Vermont tell factors but the age of young males if there's confections or not present of the flora the food and so on so the goal is to induce a slow down immune mechanism very quickly depending of a modeling condition to clear the infection as soon go back to immune status as soon as possible so the take-home message from my talk is that innate immunity is important especially in their needs and young animals for controlling pathogens in intestinal cells I've shown you that we know that dendritic cells are crucial for antigen presentation and the restoration of adaptive immune responses is in the textbook but we also showed resently that these cells are major actors of intestinal innate immunity and primary target for immune stimulation strategy host microbiota composition is not only important for intestinal immune development has been shown by band previously but then also they can modulate hospice phones during infection or immediate oral treatments as I just mentioned some specific cells so a factor can be keep for protection but also be involving adverse effect in pathology and I hope I convinced you that they're suffering all this immune mechanism it's a starting point for developing targeted strategy to strengthen immune response in the gut and of course at the end improved gut health just would like to acknowledge all the collaborators in the groups that I've done the work on Cryptosporidium anemia but also some other collaborators that work in the project for the integrity project and now especially the the people in the facilities that help us to do all these mouths young cows and also axenic chicken in the platform of FX urology thank you very much thank you have mix okay we have a few questions do you want to pick from the top so how many questions I have to answer oh you have I pick up you can pick up two or three questions it depends on the length of the answers just take the tonic receptor important to improve immune response yes they had a good target for us to manipulate diet I think we can but we need to know that this toll-like receptor are tightly regulated in the intestinal mucosa as soon as the micro flora calm for example if I'm getting maybe it depends of which toll-like receptor we are talking about for example toll-like receptor for is quickly regulated just in the very first hours after birth because of a present of the flora that increased micro RNA individual cells and in this if your cells they become quickly non-responsive to LPS of course this is one of the mechanism to avoid strong inflammatory response because of the president of a large amount of the LPS in the gut it's like Birds more resistant to parasite the differences between you bacterial pathogen compared to parasite it depends what you call Farsight if you're talking about worms it's completely different but we've and transferred in our party to the par site like Cryptosporidium for example some some of the mechanism could be similar and actually we for immuno simulation I it's something that is not yet published but we can with Newton like receptor agonist you can increase some of them make human response that are not yet present with a natural response and this mechanism also important for controlling viruses so probably yes we've entrusted our president iodide they are much in common with and Tracy are bacteria and in facility versus thank you that one question first question can use like Burt more resistant to parasite question I think this people have tried that some some the feiyu me bird for example I think they're they're more resistant to the partite that it's difficult because in I think it's a broiler industry they don't want to change much their genetics of the animals so yes probably but you need better to select what is really important and that we need to do a lot much work basic knowledge first [Music]