Scientist Stories: Valérie Verhasselt, Early Life Nutrition and Immune Development

Well, first of all, I would like to thank Nestlé Nutrition and Natalia for organizing a meeting on human milk and putting us together. Thanks a lot. Also thank Christine. I don't know if she's here because I think she managed very well all the organization, and it was really great. And I think it's a very hard task now, talking now. I think everyone is a little bit exhausted, and I will try to maintain you certainly alive and maybe interested. And I may ask you some questions, so be careful. So Sharon has asked me to talk about early life nutrition and immune development. So I have already restricted to gut immune development because otherwise it's really wide. It's already widening my topic of interest because usually I'm focusing on impact of antigen. So thanks to you, I have now done a much better review of literature on that. And I will try to see what can we take as message from the research done in mouse model. So there are beautiful studies that are done, mechanistic analysis that are done in mouse model, mainly in adult mouse model showing impact of nutrition. Very few on early life, but I think it's hard to use the data in the mouse and bring them to the human. So I will try to do that and you will see that often I will replace the mice by a beautiful lactating woman. And this is already overinterpretation. But these are more hypotheses to stimulate discussion today at 4:30 and probably earlier or later for many people here. So just to start and show you where I'm based now. I moved from Brussels to south of France where I discovered the very good quality of life on French Riviera. And then I was proposed to get a new position at UWA, now on the ocean, which is, I think, an upgrade. And University of Western Australia is wonderful campus with, of course, very well-known already researcher in the field of human milk, as the group of Peter Hartmann is there, the group of Donna Geddes now also Debbie Palmer, Susan Prescott having wonderful cohorts. So there is lots of work that can be done also with Karen Simmer on IQ. So I'm discovering the new environment and I think it will really be great. And I'm happy to get there with nice person. You will see, I know them for a long time and I'm very happy that they follow me that far. So go back, let's go to the question of today and is what is the impact of early life nutrition on gut immunity and long-term health. So why do we think that gut immunity is important for health and disease? I think it's hard also to talk to you now about immunity because many people are refractory to immunology, but I will try to make it simple because it is simple. In fact, in the gut, you have a physical barrier, the gut epithelium, which is now considered much more than a physical barrier. It's part, we can say, of the immune system because it produces molecules on both sides. To the luminal side, it produces antimicrobial peptides. It produces mucus, which of course will affect certainly the microbiota and the capacity to invade. It also produces on the luminal side molecules that will interact, communicate with the immune cell underneath. And which are those cells? Those cells are T lymphocytes that can be differentiated in many different classes with different effector function or regulatory function. In the recent years, you probably have heard about the innate lymphoid cells that just look like a lymphocyte but that do not have antigen specificity. But they react extremely quickly and have different function that are parallel to the T cell function, except that the innate lymphocytes in lymphoid cells with regulatory function have not been identified yet. Of course, you have dendritic cells that can control activation of immune system and local monomacrophages that can reinforce response that have been induced. So this is all about gut immunity. So what is— how is this gut immunity controlled? In fact, there are, as I was telling you, some factors that are produced that can be important for homeostasis when you don't have an infection, any inflammatory response. And this is mediated, for instance, by TGF-beta production by epithelial cells, IL-10 secretion by macrophage or by T lymphocytes, as well TGF-beta by lymphocytes. And those innate lymphoid cells are important also in homeostasis because they can induce the production of repair molecule by epithelium cells. And in particular, IL-22 is very important to maintain a good gut barrier. And retinoic acid production from retinal by dendritic cells, but also epithelial cell, mesenchymal cell, is also controlling immunity. And this is not only a local affair. It's something that will have impact on systemic immunity. And this is the base of oral tolerance. You eat an antigen like egg, at least the mice and probably the human. And then locally in the gut, you will induce regulatory cells that will migrate in the circulation. They will go back to the gut, but also will disseminate in the body and thereby induce systemic tolerance. So this is when everything is going well. But we also know that if you have altered immune response and inflammatory immune response in the gut, you can also have systemic and of course also local inflammatory reaction. So locally abnormal immune response in the gut, meaning less, not correctly regulated can lead to food allergies, celiac disease, inflammatory bowel disease in the colon. But also response that have been induced in the gut can have impact on cardiovascular disease, obesity. And this is mainly supposed to be mediated by metabolites produced by the bacteria. So now you know everything about gut immunity in the adult. And we go now to the early life. And of course, you know, the theory of Barker based on in utero observation epidemiological study. I think Alan yesterday also certainly insist on the importance of early life and what is early life. It may be also the life of your grandmother. That is the early life because we know more and more that there is transgenerational transfer of information. So I will mainly focus on this and mainly focus on how this period is important and how breastfeeding can help to make the child become autonomous. And I will restrict this again in the field of gut immunity. So how breast milk is impacting on gut immune development so that the neonate has a gut immune function that is not dependent anymore on the factors that breast milk that you all know are very important for mucosal immunity. And so how— what do we know about gut immune development? In fact, we know very few things. And people think they know because they're from the people— I mean, many researchers think that they will— when they analyze an adult germ-free mice, they have a neonate mice. So many researchers describe the immune system of a neonate by the analysis of an adult germ-free mice. So this has been done during years and years. And in the recent, in the last 5 years, there is now the fashion about looking at the early life and some studies are getting done. So what we know, and both in the human and in the mouse, is that the barrier at birth is not completely functional and that you have a more permeable gut in early life. In the mice also you have not well-developed crypt and villi. And what do we know about the cells in the gut? Not much. And again, many extrapolations. Everyone thinks that the neonate is susceptible to tolerance. This is because Medawa showed that the neonate can be tolerized, but this was by systemic administration of an antigen. Orally, this is not the case, and I will come back to that. So the neonate has in its mucosa, and this has been shown in the human also, in the mucosa you see many naive T cells. And you have many regulatory T cells and probably also Th2 cells. About the innate lymphoid cells, you don't know much about. You just have a few papers on one population, the ILC3. And those cells, the ILC3, are important to drive lymphoid organ development because they interact with stromal cells and this will lead to the formation of lymph nodes. So what we can conclude from studying the neonate is that there is a leaky gut barrier, there are poor effector mechanisms, and also poor tolerance mechanisms. Then there will be a maturation, and we will go on that. That leads to a full capacity to, in optimal condition, to mount specific response adapted to the situation, memory T cell and various kind of immune response. And also, of course, draining lymph node, mesenteric lymph node, but also Peyer patches where the induction of immune response is induced. So now the question of today is what is driving that maturation? So there is certainly the gene that are, of course, a developmental pathway. There is a programming, genetic programming of gut immune development. And also there is certainly a role for microbiota. Why do we think there is a role for microbiota? That's because of the use of these famous germ-free mice. You take an adult germ-free mice and you compare the immune system with a mice which has a microbiota. And then you find in those adults that if they do not have microbiota, they have increased IgE, they have a defect in tolerance, they have defect in Th1, Th17. They are very susceptible to allergic disease. So from all that, you conclude that the microbiota is important to don't regulate Th2 immune response as important to favor oral tolerance and mount very various immune response. And you also see that the size of the lymph node organs are very reduced when you don't have microbiota. So that has been done by taking an adult mice, putting back bacteria, and you see what's happening. And some research, and in particular Rick Blumberg in the U.S., has analyzed what's happening if you colonize a mice when adult or when neonate. And he found that in fact you can decrease the IgE by colonization only if colonization happens before 4 weeks of age. And this illustrates the perfect windows of opportunity that we talk about in the clinical trial, in epidemiological study. And this is really exemplified in the mice that Only during a very limited period of time bacteria can have that impact on IgE. And then the next question is how bacteria are impacting on immune development. And there is still from the same group data showing that the sugar from Bacteroides fragilis is involved in this downregulation of IgE and also NKT cells. T cells accumulation. So that's what we know about one way that microbes drive immune maturation. There are many others, but this is not the topic of today. It's certainly not my expertise. And I will talk more about how diet is impacting on gut immunity and how diet is impacting on gut immune development by acting through bacteria first. So the diet, as you all know, Many part of our diet can be metabolized by the bacteria in our gut. And what are the main factors that have been studied in the mice? So in the adult mice, again, mice can be fed with not bread, but with dietary fibers. And the works from Atahashi and others have shown that Those fibers, dietary fiber complex sugar, will be metabolized by Clostridia. And this will lead to short-chain fatty acid, propionate, butyrate, acetate. And these metabolites can act on epithelial cells, stimulate TGF-beta secretion. You remember is important for immune regulation. And also act on cells, which will lead to immune regulation. So IL-3 that will produce IL-22, important for gut repair mechanism and antimicrobial peptide secretion. Treg differentiation and also IgA secretion by B cells. And at the end, enriching the diet of adult mice with non-digestible fiber will prevent allergy and colitis. And another very well-studied metabolite produced by bacteria are the indole derivatives that are produced from— by in particular by lactobacilli that can metabolize protein-based diet containing tryptophan into those indole derivatives. And these can bind to aryl hydrocarbon receptor, also binding dioxin. And this is expressed on those famous ILC3, also called lymphocyte tissue inducer cells. And so these ILC3, as you may remember, I could have asked it, are important to develop— for the development of lymph node organ and also for the production of cytokine involved in tissue repair. So we see that the bacteria colonizing an adult can act by molecule in the surface, can act by metabolizing diet. In particular, what has been studied much is metabolism of fibers and metabolism of protein. Leading to short-chain fatty acid and endo-derivative. And so now let's go to the neonates. So for the ones that are not familiar, this is a 1-day-old neonate. Looks like a preterm, but is already very dynamic. And however, he will not receive bread. No protein-based diet. Has no bacteria or very few bacteria in its gut. How can you extrapolate what is known from an adult mice to this neonate? And of course the answer and the magic come from human milk. So breast milk is even more magic than I thought before when I prepared all that. Because we don't— a neonate doesn't eat bread. In fact, the mother will make a surrogate, I think, of dietary fibers. In the breast milk, which are the oligosaccharides. And those human milk oligosaccharides can be transformed by the Bifidobacteria into short-chain fatty acids. So although a neonate is not supposed to receive non-digestible fiber, it has, however, the capacity to receive short-chain fatty acids because the mother will make a surrogate for it. And also, the neonate has no— no, not developed microbiota at birth, but as I think Erika will discuss after, mother milk is bringing its own microbiota and immunomodular factor and antimicrobial factor that will affect the bacteria and certainly will favor the differentiation and the growth of those bacteria. And so that's one way. And for the indole derivative, there has been a science paper published by Gomez de Agüero from the Macrossan group, showing that in fact the maternal microbiota in the gut will produce some indole derivative that will be transmitted to the breast milk. And so the baby also received indole derivative coming from the mother microbiota. So in addition, the IgE, IgA, and IgD from the mother are important for transferring this indole derivative and ensure the function of activating IL-3. So I don't know if you find that magic, but I find that really magic. And so after this little thing, now we come more to the research my team has been conducting for 10 years. Is how the diet can directly impact on immune development and gut immune development. So instead of going through metabolization by bacteria, can the diet directly impact? And I will focus on certain molecule, will not review everything. So I don't want you to die. So, and this is not my expertise, certainly. So the cruciferous Some vegetables like broccoli, cabbage contain indole derivatives that can, in absence of bacteria, have the same role and stimulate HLA receptor. And as you remember, prevent some infection and inflammatory response in the gut. And also, we heard this morning from Artur Moreau Vitamin A is important. It's found in breast milk. And but here in the adult, there has been many, many work in the recent year on impact of vitamin A on gut immune regulation. And what we can summarize is that vitamin A given orally is important for T-cell homing and B-cell homing. So the T-cell that activate in the lymph node, they have to go back to the gut to exert their function. This is dependent on vitamin A. Vitamin A is also very important to amplify immune response, either regulatory immune response but also inflammatory immune response such as T helper 17. And vitamin A is also very important again for this famous ILC-3. And if you don't have vitamin A in the diet of an adult mice, they have much more, much much less LC3 and much more LC2 and are very susceptible to allergy. So that's still in the adult. And a more recent paper also published in Science analyzed the role of antigen, dietary antigen. So you know that you have million, billion of bacteria in the colon, also in the small intestine. The small intestine is certainly exposed to another source of antigen. These are the one coming from the diet. So with the mice, it's nice. You can do a very precise thing. So they took mice that have— that are germ-free, so they have no microbial antigen. And they also took other mice that have no bacteria and in addition have an amino acid diet. So they have no intact protein. They check the nutrient composition is correct. And so those mice are called antigen-free. So they have no microbiota-derived antigen, no diet-derived antigen. And by combining these analyses, they found that the antigen arriving from the food in the gut are important for the pool of memory T cells and for regulatory T cells in the small intestine. So germ-free mice— well, bacteria is important for Treg in the colon.. And dietary antigen are important for regulatory T cells in the small intestine. And certainly you understand the importance of this in the context of children that have allergic disease, severe allergic disease, and sometimes they are given amino acid formula. So on the short term, this may be good, but in the long term, this may really impact on the capacity of the gut immunity. To have regulatory immune response. So this is a comment. So I mean, antigen derived from the diet are important because they increase the pool of regulatory T cells and the tone of regulation in the small intestine. And probably we could also refer to a study published by the Pasteur Group showing that that diversification of the diet in the first year is associated with more regulatory cells and prevention of allergy. This may be something that is a parallel to what is found in the mice. And now what about the specific antigen? This certainly you all know that having introduction of food antigen by oral route oral route can induce antigen-specific tolerance, meaning that if you eat egg at certain period of your life, probably around 4-6 months or 4-11 months for peanuts according to some studies that have been recently review— meta-analyzed in the paper of JAMA, show that you can induce antigen-specific tolerance. So you eat egg, you're protected from egg allergy, not from a fish or other allergy. So it's a very specific prevention of food allergy by giving oral antigen in a certain period of life, at least a period when you eat already solid food or you're capable to eat it. And studies in the mice show that, okay, it's good to expose orally. It may be good to expose orally to induce prevention. But this may not be sufficient. And in particular, the group of McKenzie showing that having short-chain— having vitamin A can help to induce oral tolerance and antigen-specific prevention of food allergy. And another group saw also that short-chain fatty acid produced by bacteria also helps. So of course, we also always start by a very specific question, and then you can see that you can improve by adding other molecules such as metabolites produced by bacteria or vitamin, like vitamin A. Again, what's going on with this little pup which is not supposed to be exposed to solid food, and how will it have a maturation of its— and impact on its gut immune function while it's not receiving solid food? And of course the question goes to the breast milk. So in utero here I would like to mention a study performed also in the mice showing that retinoic acid and vitamin A nutrition in the mother is important for lymphoid organ development and that vitamin A is really critical to drive the ILC3 development as well as stromal cell organizes cells secretion of cytokine that will be critical for lymphoid organ formation. And now about the diet-derived antigen in early life. So how are you? You okay? Yeah? Okay. So I will have a drink. So to summarize, we have seen that microbiota can directly impact on gut immune development. Few studies in the neonate. It can act because microbiota metabolize some food. In particular, it can produce indole derivatives and short-chain fatty acids. Antigen in the diet have an impact on the regulatory tone. And this is shown in adults. We saw that the mother microbiota can produce indole derivative and also can, thanks to HMO, can give short-chain fatty acid to the neonate. And so let's go now to the focus of research of the team is how can we induce oral tolerance, antigen-specific oral tolerance in a neonate. So as I was telling you just As I said before, if you give antigen, which may be a little bit surprising, but if you think it's something that you could envisage, it's giving directly egg antigen to a 1-day-old mice. So people have thought this may be something that can be done. So they test it and they find that it has no impact on oral tolerance. It can even prime the immune system. So you certainly understand this is not physiological at all for a neonate to receive solid food at day 1. And you see that the immune system is not ready to cope with a high amount of antigen given that way. So we developed 10 years ago now a model where we gave a food antigen to a lactating mother.. And we measure the level of antigen in breast milk. And it was very low. It was in the range of 1 nanogram per ml. And you know that beta-lactoglobulin, for instance, in cow milk is in the range of milligram per ml. So we are absolutely not in the same range when we deal with exposure to antigen in a breastfed neonate. And we wait that the mice is 6 weeks old. So that's nice compared to the human. You only wait 6 weeks. And then you check, we check the allergic disease susceptibility, either respiratory or food allergy susceptibility, to see the impact on local immunity and systemic immunity. And this is something published now a while ago, but I will just briefly come back to it. What we found is that you can induce oral tolerance, prevent allergic disease systemically By giving to a mice, not a mother, not a human, we found it in the mice. By giving egg antigen to a lactating mother, you can prevent allergy. But the allergen only is not sufficient. And here comes the breast milk. You need the TGF-beta from the breast milk to ensure that tolerance induction. So while in the adult, just giving again egg antigen can induce tolerance, in the neonate, you need the cofactor from the breast milk to have lacturance induction. And this illustrates this unit that in fact the mother milk is complementary and complements the so-called immunodeficient immune system of the neonate as otherwise it can't mount oral tolerance. And we next wonder whether the maternal immune status can impact on oral tolerance induction. Just to mimic the fact that you have allergic or non-allergic mother. Would it make a difference? And so Eric Mosconi was a PhD student analyzing this. So this is the initial model. We found that antigen, anti-GF-beta could induce tolerance. And a few years later, we were capable to show that if the mother is immunized, you then have immunoglobulin in the milk, binding to the antigen, and those immune complexes are now very well captured by the epithelial cell. The transport of antigen is protected, and you have very strong protection mediated by FOXP3 Tregs that are known to be important for tolerance. What was also interesting that it took a few years to understand in this case what kind of cell were mediating tolerance, and it was not FOXP3 Treg but Th1. Cells. So this illustrates that by different ways you reach tolerance, not always the same kind of tolerance, but milk can by different ways achieve the same goal by different ways. So if you have antibody, you have FoxP3 to erect tolerance. If you don't have antibody, you also have tolerance, but by Th1 mechanism. And the more recent work by Mathilde Tuffcoyer was to check whether you— there is an optimal period to expose the breastfed neonate to an antigen to induce protection. So what we did is that the mother was exposed to egg antigen during the first week of lactation, the second week of lactation, or the third week of lactation. And we check if the protection, the prevention of allergy was the same. And you can see here that when mice were breastfed by exposed to mother during the first week of life or the second week of life or the third week of life to egg, and you assess when they're adult allergic inflammation in the airways, you see that we can prevent respiratory allergy by giving egg antigen to lactating mother only if the mother receive it in late gestational period. In the very first period, in the beginning of lactation, early exposure is not working. Although you have TGF-beta and other factors important for TLRN. So we thought it may be not a problem of the breast milk, but a problem of the neonate in the first week. So we assessed the gut barrier of a neonate. And as others have shown, we found that the neonate gut barrier was leaky. And you can see here the in vivo transfer of a protein across the gut. And we measure it in the serum. And we saw that when you give an antigen during the first week, you find it at much higher level at week 1 than if you give it at week 3. We also found that the neonate had— neonate means we analyze at day 4.. And we analyzed at day 17 or 15. We found that if you look at dendritic cells in the lamina propria, which are important for production of retinoic acid, as you remember, very important to drive immune response in the gut, the neonate has a poor capacity to metabolize vitamin A into retinoic acid. And we also find that the dendritic cells from a neonate from the mesenteric lymph node were fully capable to stimulate T-cell proliferation and differentiation. And so why is that so? We will come to the vitamin A. In fact, we measured the level of retinol in the serum of mice that were 4 days old or 15 days old. And we found that the level of vitamin A in the neonatal mice was very low. So I don't know if it was very low. I found those values, so I didn't know if that's very low or not low. So I asked people of the biochemistry lab of the hospital, and this is a region— that's all that has been done in Nice, was I was working there. And in Nice we eat very well, and I asked what are the values that are accepted for the lab value that you accept for retinal level in the serum. And here is the curve I received. And you can see that the minimal value that are accepted are considered as normal, very low at birth and in preterm, as you certainly know, even lower. So the value we find here in the day 4 mice quite comparable to the one you find in a human neonate. So it looks like it's a conserved observation that you have low vitamin A in the serum of a neonate. And we can discuss this. And then you will find many other papers showing that. So we wanted to further investigate the role of vitamin A. And we supplemented the mother, which were, however, fully had a very healthy diet with enough vitamin A, but we gave them extra vitamin A. And this way we could increase the retinal level in the serum of our day-4-old mice. And then in fact we accelerate the maturation of the immune system of the neonate. We found that those mice receiving vitamin A through the breast milk had a stronger gut barrier already at day 4, more developed crypt, the dendritic cells were not capable to metabolize vitamin A. And the DC dendritic cells were also capable to activate the immune system. So to summarize this with a scheme, we found that in the mice, the neonatal mice has physiologically low vitamin A level. And this is associated with the leaky barrier. And it's also associated with the poor capacity of dendritic cells to activate T cells. And in 3-week-old mice, they have in fact received vitamin A through breast milk. So now the level of vitamin A in the serum is normal compared to the adult. And in that case, you find that the gut barrier is now much more strong and dendritic cell much better to activate. And if we give supplement of vitamin A to neonate through breast milk, we found that we have now a neonate that is aged and comparable to a 3-week-old mice and now also capable to mount tolerance, be protected. So from all that, we come with some hypotheses. And we heard this morning that vitamin A deficiency is a serious public health issue. I would like to propose the idea that vitamin A deficiency, the physiological deficiency, so the normal value for a neonate but which are low compared to an adult, may be a contributing factor for allergic disease in Western countries. And so there is one paper analyzing this in Finland. And it's not an intervention study. Publication looking at association between level of retinol during the first months of life and the risk of atopic sensitization. And they find an association between low level of vitamin A and risk of allergy. Okay, so that's in Finland. But if you go to Guinea-Bissau, there has been an interventional study where neonates are supplemented at birth with 50,000 units of vitamin A. And risk of allergic disease is evaluated at year 10. And they find that in the female only, giving supplement of vitamin A was increasing the risk of allergy. So how do you reconcile our mouse study, the clinical observation in Finland, and the one in Guinea-Bissau? I think a major difference is that in Guinea-Bissau, you have endemic enteric infection. So the gut mucosa is inflamed. So all the immune response are going in the direction of inflammation. And now you give, you wanna induce tolerance and you give vitamin A. In fact, you will not induce tolerance, you will promote inflammatory immune response. So this is a hypothesis I would be happy to test. Vitamin A supplementation in a country with not endemic infectious disease to test whether it could help. So how much time do I have? Like 5 minutes? 5 minutes? And so, so to summarize, we found that again the breast milk accomplished what I would say magic function because it will bring dietary antigen to the neonate. And in addition, it will bring factors that complement the deficient immune system of the neonate. And in addition, it brings factor that will mature it and at the end allow allergy prevention, at least regulatory function. I think I will pass this. And just to summarize what we we know a lot about is that breast milk can prevent infectious disease. And this is certainly because breast milk is bringing mucosal passive immunity. Our studies and also the studies from Ima Pentilla show that breast milk brings immunoregulation and also with oligosaccharides. But we also find that in addition to bacteria that can promote gut immune maturation, vitamin A is important. To favor immune development. We found that antigen transfer through breast milk can educate the immune system at a certain period of time. And vitamin A can help in that education. And this can lead to immune tolerance. And the 2, 3 last minutes, I will talk then to you about the research we've been doing now in the last 4 years on house dust mites. And I will go to the take-home message. So house dust mites, why should I talk about house dust mites when I talk to you about early life nutrition? That's because in fact we— I will, if you want, I will explain why I came to that hypothesis. But I came to the hypothesis that house dust mites may be present in in human breast milk. And indeed, that was the case. And I skip all that. And as you can see, if you analyze the presence of an allergen of house dust mite, the P1, you can see around 100 picograms per ml of house dust mite allergen, the P1, in colostrum from Brazil, France, Australia, and also in more mature milk. And this is found in comparable level to an egg antigen. So yes, you can find a respiratory antigen in the milk. And in fact, we found that it was also present in digestive fluid of healthy people. So derp is in fact mainly— house dust mite is mainly ingested and doesn't go to the lung because these are big particles. So they are mainly going to the gut. And I think we should consider it as a dietary antigen to see impact on gut immunity. And this is what we have been doing. And just to summarize, we find that mice exposed to house dust mite during lactation period were in fact more susceptible to allergic disease. And this was shown for the lung infection, inflammation. So mice receiving house dust mite through breast milk are more susceptible now to respiratory allergy. We found that egg was preventing allergy. We found that having house dust mite in breast milk is promoting allergy. So this is very bad news. And it's not only a study in mice because we did now association study looking at level of house dust mite antigen in human milk and risk of respiratory allergy in 5-year-old children and atopic sensitization. And we find that having high amount of DERP, I mean high is 50 picograms per ml. So I think homeopathic has a big career with human milk because it's really important to see the level of antigen that can prime immune system for long term. So we found it was associated with higher risk of respiratory allergies. This is for the next time I'm invited. And so to conclude, just look at this. We found in the mice, and we have some evidence in the human, that giving an allergen by oral route can be a route for sensitization. And it's not only inducing tolerance. So we now have moved the mind in the, okay, give everything by oral route to induce tolerance. No. Certainly not with house dust mite and maybe other allergens. And this also can maybe taken into account to think about other factors that are modifying gut immunity, that are the bacteria, that are the dietary antigen. And there is also house dust mite that has been with us for million years. And I join again Alan. We had house dust mite with us And it's important. It's important for sustainable development. It recycles our skin, our nails. It's important. And we survived very well, but in the last 30 years, we don't live very well anymore with house dust mites. So something has changed. And so that's one of the last— yes?— slide. This you know. And you know also this. You know that. And we also know now that breast milk is that magic because it can induce whatever you want. It can induce tolerance. It can induce priming. And you may also find this a bad thing for prevention of allergy. But of course, we ambitious and we want to counteract that. But we would also like to exploit this and think that, okay, you can stimulate immune response orally by having an antigen in breast milk. So of course we propose that we may maybe vaccinate children through breast milk. This is something I hope to do in the next 10 years. And for prevention of allergy, we have identified a factor that's not for today, but I hope it will be published soon. We find that gut, at least how the smites in the gut modify immunity has long-term impact And now we are looking at how to counteract that by giving intervention to the mother or to the neonate. And then that was the talk of today. And here are the summary. But I will leave just you remember breast milk is bringing a bread to the neonate, allowing him to have short-chain fatty acids. Which probably have an impact, but this has not been studied. Breast milk is bringing in toll derivatives that when you are older, you can get from bacteria or from your solid food. Dietary antigens are important because they give a regulatory tone and in addition can induce antigen-specific protection. And breast milk is bringing factors that will increase this possibility. That's for some antigen, but not for all antigen, and in particular house the spike is priming the immune system. And I thank you a lot for staying alive.

About 10 minutes for questions.

Yes.

Thank you, thank you, Valerie, for your wonderful presentation. So you talked about allergy exposure in the lactating period. So can it be done in the early pregnancy? Because we know the critical period of pregnancy when the fetal genome is programmed. And all we know that maternal diet influences the newborn's or the following baby's food habit. So there is some experiments giving mother a carrot juice throughout the pregnancy. It was seen that during weaning, the baby shows preference to carrot. So if we expose the mother in a low dose to allergen in the early pregnancy, does it work better than the lactating period?

Well, as I say, we unhappily, we have to focus because each question takes like 3, 4 years to be answered. So I haven't looked at pregnancy, but I know that you find dietary antigen in amniotic fluid. And also house smite antigen have been found in amniotic fluid. This was published in The Lancet in the year 2000. So I think that indeed, certainly in the human, which has already an immune system more mature in utero, can probably certainly be already influenced by this. But in all the experiments we did, we take naive pups, so we exclude all what did happen in utero, exposed only through breast milk to the antigen. So I totally exclude your question. I'm sorry.

So I have a question in terms of— you spoke a lot about the sort of metabolic products, but you didn't really talk about the microbiome itself. And maybe we'll hear a little bit more about that, but in terms of direct interactions, for example, we know Bifidobacterium Bifidobacteria has been shown to increase IL-10 expression.

So— Yeah, well, this is not my expertise, but I think it can go through the metabolites, because how do you explain Bifidobacteria induce IL-10? So some people have shown, for instance, that polysaccharides from Bacteroides is capable to induce IL-10 and stimulate Tregs directly. Others show that it goes to short-chain fatty acid that induce those, that TGF-beta. But I think currently I'm not doing that much on microbiota. But certainly, of course, it interacts. And having that, you saw in the adult model, it has been shown that having Clostridia can help in inducing tolerance, probably because it brings those short-chain fatty acid that bring IL-10 or TGF. So yes, it's not one factor that is magic.

Bruce? So how generalizable is the dust mite phenomenon? So that was perfectly benign for a million years, you say, and now it's a problem. Is it specific to dust mites, or virtually all of the antigens that were benign before are now priming at the level you said?

Yeah, what we can say is we find that egg antigen is not priming, it's inducing tolerance. We know that house dust mite is priming. And we know that if we inactivate the protease from house dust mite, in that case, we don't have protection, but at least we don't have priming. So we identifying the factors in house dust mite that are contributing. So then you wonder why the protease of house dust mite in picogram per ml are activating the immune system while you are eating protease and that have no impact. So this is really a mystery. But at least we know that having protease inhibitor could counteract that. And maybe other way that are more— that you may— we also looking at how to modulate the bioavailability of that house— the mother is exposed, it goes through the milk. How do you interfere with all this transfer? How do you control it? What I can tell you is there is absolutely no relation between the environmental exposure and the level in breast milk. So that's That's very important because after the publication, I saw on blogs mothers saying, oh, we should take care of house mites. I saw it's dangerous. No, no, no. So I'm happy to publish as soon as possible that they can keep house mites around them. It doesn't change anything. Yeah, we don't live anymore well with them. I don't know why, but that's the way it is.

It was a lovely talk, really. [Speaker] That's really great. So I have two different questions. And I don't want to be too picky about this, so I'm just going to ask the first one and see if there's time, there's a second one. So the first one is more of implications of the basic science and clinical study design and from there public health interventions. And I just want to probe more to understand where you think the science and public health are intersecting. So as you know, exclusive breastfeeding has been the standard from WHO and so on. There are those who are in allergy who are then proposing this should be the standardized, as a trial that was done in Britain, standardized introduction of allergens to the baby directly between 4 to 6, which would change that public health recommendation. Or there's the possibility, right, in other studies, yours and others, to give it to the allergen by consumption from the mother and then it's conveyed through milk. So do you have any sense of really how the relative effectiveness, the relative appropriateness of these different strategies? And also with something like egg or peanut or something, you know, is there any variation in these allergens and the way from place to place that that would matter?

What I can say from the studies I have conducted in mice and what I have read is that it's hard to induce tolerance in a neonate. Very hard. So even people, the group of Weiner looking at prevention of EAE, he tried to prevent EAE in rats by the same protocol and he could not in a neonate. I showed for egg, but it's the same for EAE. So it's hard to induce tolerance. And we gave egg and we modified the amount. We were not capable to induce it in the first week of life. So that's one thing. So it means that very early on, doesn't matter if the mother is exposed or not. It's probably, according to that in the mice, later on that it's important that it's exposed through breast milk. Well, it doesn't do anything in the first week. So maybe it would correspond to 4 to 6 months period in the lactating mother. It's at that time that she should be exposed. And I have now data with Susan Prescott and Debbie Palmer that show indeed that at 4, 6 months, having egg antigen is associated with protection. But we don't have enough N, a number of cases. It goes in that direction. Now for how's the smile? This is another thing. It works very well from first week of life. You can activate the system. And well, I think it's again evolutionary thing. It's allergy and tolerance is not a problem for survival. What is a problem for survival is effector mechanism. And so it's probably important that the neonate in case of a danger, so now how's the house dust mite is considered as a danger by a neonate, can prime the immune system. And we see that at week 1, they are really primed. So I would suggest that the mother eats what she wants during the first months. It would not change anything. And the late phase of lactation, it could induce tolerance as long as you don't have too much house dust mite in your breast milk. Thank you.

Yeah, so— good, thank you. So I'm very interested in pursuing the interest in the FUT2 gene and its upregulation and what's understood, and you were kind of at the edge of some of that mechanism, IL-22 expression through ILC-3 and the microbiome or specific microbes. You're really alluding to here lactobacilli.

So—

Yeah, that's what I am. Some of the question really has to do with how specific is it to lactobacilli? Is this really more a set of a variety of microbes that this is one example of?

Yeah, that's what I— this is not something I have done. This is something I have read that Lactobacillus is particularly capable to metabolize tryptophan, but maybe the microbiota expert can tell if other bacteria species can do it. I certainly read that this is one bacteria that is capable and shown to be effective in that metabolism.

That's very helpful. Thank you.

Okay, well, thank you very much. Thank you.