 Good morning. I'd like to thank Lita for the invitation to come here today and talk to you about our work and how it relates with our HMP demonstration project on atopic dermatitis. We've heard a lot about the gut microbiome and I think it was nicely organized that Susan's talk about the skin and wound healing segues very well into my focus on the skin microbiome. And one of the overarching questions that has developed in our time in looking at the skin microbiome is, do skin microbes influence host skin immunity? I'm going to give some background for those who are not familiar with the skin microbiome work in order to be able to talk about the microbiome and examinitis skin. I'm highlighting here a series of experiments by a collaborator, Yasmin Belcade's group, to highlight some of the differences that we see in that this not only is the skin microbiome different than other body sites, but that the skin immunity and the skin microbial relationship is also distinct. Here, Shruti, who was a graduate student in Yasmin's lab, she treated SPF mice. These are all experiments in mice with antibiotics. And what we can see in the gut that after antibiotic treatment, there's a significant shift in the microbial communities, but you don't see those significant changes in the skin microbial communities. Also, Shruti looked at the immune cells that were in the gut, IL-17MA, as well as interferon gamma producing cells in the gut. And as compared to the skin, once there was treatment with antibiotics, there was a significant reduction in the interferon gamma and IL-17A producing cells. So, in distinct epithelia, there are differential responses to antibiotic treatment, at least in these mice. So, Shruti then went on and in comparing SPF mice and germ-free mice, SPF mice here, germ-free mice looking at IL-17A producing T cells in the gut and the skin, we see here that there are reduced IL-17A producing T cells in the germ-free gut and skin. However, what she then did was topically mono-associated stethepodermidis in these germ-free mice. And interestingly, there were no changes in the interferon IL-17A producing T cells in the gut. However, in the skin, there was an increase of IL-17A producing T cells in the skin after application of stethepodermidis. So we can see here that skin microbes do influence the immune system, but is there a function? Is there a biological result when you do that? So what then Shruti did was use a Leishmanian major infection model. She infected the ears of these SPF mice and germ-free mice. In comparing these two, you see that there is a local cutaneous response in the SPF mice as compared much larger than the germ-free mice, but also you see increased interferon gamma producing T cells in the SPF mice as compared to the germ-free mice. So then when she topically associated stethepodermidis in these germ-free mice, you see an increase in the local cutaneous response as well as interferon gamma producing T cells that are in the ear. And these resemble what you see in the SPF mice, but also here we see in parasite burden that you have a decrease in the parasite burden when you treat or topically associate stethepodermidis. So skin commensals can restore immunity to Leishmanian major infection in germ-free mice. So summarizing this part, the host immunity and microbial interactions in skin are distinct. In mice, skin microbes can tune the level of activation and function of skin-resonant T cells, promote immunity to pathogens, and drive responses locally that are distinct and independent from the gut flora. But what about human skin immunity and microbial interactions? Unfortunately, we don't have a lot of germ-free humans walking around that we can do these types of experiments, but what we have done is started to look at what are the microbes that are on human skin. So again, this is a background for those who are not as familiar with human skin microbiome surveys. These are highly selected due to limited time that I'm not going to go into a lot of all the surveys that have been done. You've seen this figure before, but the reason why I'm showing it again is I want to emphasize that in skin here in pink there is a wide variation in the microbiome that you see in skin as compared to the other body sites. And that is because the skin surface is highly heterogeneous. And that is one of the reasons why in this study we selected 20 skin sites. These are distinct microenvironments on the skin surface, but I also selected them because they were sites of predilection for skin disease, specific skin diseases for which microbes have been postulated to potentially have a role. And David Relman showed this figure earlier, so I won't go into it in detail. But relative abundance of predominant bacteria appear to be dependent on the microenvironment. So we've talked about this throughout yesterday and some this morning, how there are carefully defined cohorts that we have looked at. Most of these are adults. We've talked about what happens. There have been the studies by Maria Gloria looking at the neonatal period, but what happens after that? Ruth Lay talked about at least for the gut microbiome how that can transition in the first two and a half years. But what about the skin? We don't know that information. In the other next transition, puberty. This is a turbulent period of time for our bodies. What happens between as we transition into adulthood? There's something called a tanner stage. For those of you who are not clinicians, a tanner stage is used by pediatricians to assess the level of sexual maturation in an individual child. This is based on the development of breast tissue as well as genitalia, cubic hair, but it ranges from one to five, one being pre-puberty, five being fully developed adult. So what we have here is we had cohorts of patients that were ranging from two to adulthood. So what Julia O. did in our in Julie Segre's lab was take these data and compare them with each other. And what based on tanner stages one, two, three, four, and five, we see the striking difference between tanner stages one, two, three, and tanner stages four and five. This is from the nary's, but we see a similar separation in other body site, in other skin sites. What drives that? Here we see a relative abundance chart, which you've seen a lot in these talks leading up till now. And here is tanner stages one, two, three, and then here is four and five. I do acknowledge that the ends are pretty low in these populations. But what we see in tanner stages one, two, three, that we have increased proteobacteria and more lipophilic bacteria in tanner stages four and five. That makes sense for everybody who's gone through puberty. You recognize that your body is changing dramatically. And one of the things that changes is the further maturation of our sebaceous glands or oil glands in our skin. So it's very possible this may be one of the reasons why we have more lipophilic bacteria, because they can thrive in that type of environment. So we've talked a lot about 16S taxonomic surveys. We've heard some about the Virome and several of the investigators looking at that. But what about the fungal microbiome? So more recently we've published a project looking at sequencing the fungal organisms on the human skin surface. But in order to do this, we had to go back from the beginning and figure out how do we optimize sample collection. And this refers back to the conversation we had in the open force session yesterday. What are the protocols to use? Optimizing DNA extraction, we had to deviate from the standard protocols for extracting DNA in order to optimize extraction of fungal DNA using bead beating, opening the fungal cell walls. What primers would we use? There are individuals who use 18S, some who use 28S. There's the ITS region here, ITS1, ITS2, that stands for internal transcribed spacer region. And so based on our analyses, for us at least for skin, that ITS1 provides more taxonomic resolution. This alludes back to Jean-Clavel's comment yesterday that just because it works for the gut or for the vagina, it may not work for the skin. And so this is for at least in our experience, ITS1 worked better for taxonomic resolution. So what we did was we had 10 healthy volunteers. I selected 14 sites. These are not identical to the ones that we did in the bacterial survey because these are, again, sites that reflect sites of predilection for skin diseases for which fungal contribution may be playing a role. So in 10 healthy volunteers we see here. Then we, if you look at the relative abundance charts, each of these horizontal lines represent one body site. Each of the vertical bars represents one of the healthy volunteers. And one of the most striking things that you can observe here is that malicezia predominates in 11 out of the 14 sites. That's the purple. That's why there's so much purple. But on the heel, toenail, and toe web space, so on the feet, there is much greater diversity. We have fungi everywhere, but it's just a different population depending on where you are. One of the things that you may notice is that healthy volunteer 7 is a bit different. This individual, again, talking about protocol standardization, our eligibility was no one could take, have taken an oral, antifungal, oral antibiotic within six months of being sampled for this protocol. And this individual had completed a course of oral antifungal seven months prior to being sampled. And so it's not clear whether the differences we see are related to residual effects of taking an oral antifungal or whether or not the person's predisposition, they were taking it for a toenail infection, whether a predisposition for a toenail infection represents some difference in their fungal microbial communities. Interestingly, I don't have the data here, but their 16S survey for this particular age, individual HV7, was similar to the other. So the bacterial microbiome was resembled any other healthy volunteer. But it was frustrating to see so much purple. There's a lot of malassezia, that's not that helpful. But one of the questions we had was, what happens if you speciate? Getting down to the speciation level, how do we do that? Well, we couldn't do that based on the databases that were available. So we had to cultivate our own malassezia and do genomic sequencing to actually populate the databases for us to be able to speciate. So when you do that, you again see there is striking site specificity for skin. Sarkis was talking about how there's specificity in the gut, but also we see that for the skin with regards to the fungal microbiome as well. So we can see in this area here, the external ear canal, behind the ear, the forehead, that malassezia restrictor predominates. But in other sites, we have the upper back, the back of the scalp, the inside hip, those are predominant, but you have malassezia globosa. So even though they're all malassezia, there are specific species that we're seeing at these different body sites. So I did mention that we did 16S. So from these same clinical samples, we did ITS, one sequencing as well as 16S sequencing from these samples. And what you see here is interesting anatomically in that those central body sites from the head and neck or core body sites listed here, they have a relatively limited number of growth guards to reach richness, relatively limited numbers of different types of bacteria and fungi. Whereas on the arms, here's the palm, the forearm and the inside elbow, there is a higher richness, a greater richness with regards to bacteria, but still relatively limited for fungi. That's different for the feet here, the heel, toe, ebb and toenail, relatively limited richness for bacteria, but a much greater richness for fungi. So there are these regional differences that cannot be explained just by sebaceous, moist and dry and that it's going to require a lot more understanding about human physiology and skin physiology to be able to explain these differences. So just summarizing this beginning portion, the skin bacterial microbiome is highly dependent on the sampled skin site. The neonatal skin bacterial microbiome varies based on mode of delivery, we've talked about that, that there are dramatically major shifts that we see in the tanner stages 1, 2, 3 versus tanner stages 4 and 5 and that fungal communities over the skin surface differentially vary from the bacterial microbiome. Now I'm going to shift into what my charge was into talking about eczema to skin, specifically atopic dermatitis and then briefly I'll talk about some data from other groups and our group about primary immunodeficiency syndromes and why that's interesting. So for background, those who are not familiar with atopic dermatitis, it's a chronic itchy inflammatory skin condition. It is not considered an infectious disease, it's an inflammatory skin condition, yet these individuals respond relatively well to antimicrobials. So there is something going on that suggests a role of microbes. It affects 15 percent of U.S. children at a high cost financially as well as socially. The quality of life in these children and in these families is severely adversely affected. And I mentioned before there is this association with microbes we observe and when we take care of these patients that disease flares are associated with increased colonization and infections with staph aureus, but also some a subset of these patients are at high risk for severe spread of herpes simplex virus infections and if they come into contact with small pox vaccinees. There is something that's been termed the atopic march in that there are 40 to 70 percent of those with severe atopic dermatitis over time go on to develop asthma and then hay fever. So these three diseases are termed the atopic triad. The incidence of these atopic diseases have doubled in the last three decades in industrialized countries suggesting there may be a possible external factor and this alludes to some of the comments yesterday by Marty and again today that it is unlikely that our human genome could change in that period of time and that an external possibly microbial contribution may be playing a role. Interestingly in mice and murine studies skin exposure to antigens can result in subsequent mucosal sensitization to those antigens suggesting that if we could somehow modify what happens in atopic dermatitis could we then go on and abrogate the development or the the disease severity of asthma which has significant morbidity and potential mortality as well as hay fever. So understanding the trigger triggers of atopic dermatitis may allow us to modify the development of AD and atopic disorders and potentially develop therapeutic targets. So atopic dermatitis is a complex disease it looks very simple here but this is not the whole story but just emphasizing we've talked about barrier the gut barrier earlier today but emphasizing I won't talk about it here but it is important in atopic dermatitis the skin barrier we know in um if you have a mutation in the skin epidermal protein valagran it's highly associated with the development of atopic dermatitis a particularly the kind that goes on to develop asthma and hay fever. We know the immune system is deranged in these individuals in that they have extremely high IGE levels and antimicrobial peptides in the skin are reduced but and we've also talked about the microbes and that is just one component of this complex disease. So our study we recruited pediatric patients with moderate to severe disease and healthy age match controls. I sampled them in characteristically affected sites as I mentioned before dermatologic diseases have sites of predilection where we find that and that helps us in the diagnosis of these diseases but it typically occurs in the anticoabital FOS and the popliteal FOS of that being the inside elbow and behind the knee. We selected the volar form or the inner form as a control site because it's less often affected in in moderate disease and it's an adjacent site. We also sampled the nares because that is a site of carriage for staff aureus and I sampled them during the baseline flare and post flare time points and these are just SCORAD scores which is a method for assessing severity in these patients and you can see that over time their SCORAD increases during the flare and decreases after they've been treated. This figure just demonstrates that when you have more severe disease that we observe a decrease in the bacterial skin communities but that drop in diversity is not everywhere it is very site specific again it is at the sites where we see disease appear the inside elbow and behind the knee but not at all time points this in particular are disease flares these are the natural true natural history of the disease where they have not been putting anything on their skin these the the blue flares are individuals who put on topical steroids potentially two days before seeing us or sometime within the seven days before they were sampled so these flares are truly the natural history of the disease but what are the bacteria that seem to be driving this decrease in diversity so looking at the genus down to the genus level here are the healthy controls these are the baseline time points for the atopic dermatitis the flare time points the no treatment ones versus the intermittently treated ones and the post flare time point and what you can see is that there's a dramatic increase a significant increase in the pink the staph locoxi in the skin of these patients during a flare but we do see increased staff in in proportion or relative relative increase in some of these individuals so that was quite a concern again we had to go down to speciation level because there's staph epidermis staph aureus staph epidermis again is a known skin commensal staph aureus is a fairly common pathogen and so it was important to us at least from the genome level they're very similar so we had to know which one was which because it makes a difference at least clinically and so what we did if you focus primarily on the pink the staph locoxi and we speciated those use it was reassuring to see that most of the staff we see in the healthy controls those are staph epidermis staph hominus known skin commensals but what we do observe is yes there is an increase in the staph aureus that we see even at baseline but it is a dramatically increased during the flare the natural history of the disease we see increased staph aureus but also staph epidermis that was not something we did expect to see but there is that is one of the benefit of looking at the whole microbial community with regards to disease progression and then it decreases once patients have been treated and one of the questions people often ask well you gave them antibiotics but often these majority of these patients were limited their treatment was limited to topical steroids or some of them do take dilute bleach baths which is like having a small swimming pool in your own tub so that's what we observed but what is what remains a question is this this our correlations what happens to get from this point to this point or what can what can we do so that these you never you remain a control you don't go on to develop atopic dermatitis those are questions that remain we did look at the fungal communities on the skin Julia oh looked at this these are baseline flare and close flare for a few of our patients who had atopic dermatitis and although you see fluctuations in the bacteria we don't see as this that type of fluctuation in the fungal communities on the skin so i'm going to shift gears a little bit and talk about primary immunodeficiencies there are some cohorts of primary immunodeficiency patients who have eczema to skin disease and the benefits here are they genotypically have the same mutation they are monogenic disorders and some of these patients have atopic dermatitis like skin eruptions and these eruptions can be antibiotic responsive again and so that was one of the reasons why we pursued these cohorts and asking do common and rare diseases with similar clinical phenotypes do they share skin microbiome features and how does the innate and the adaptive immunity shape the skin microbiome we haven't been able to ask all of these questions but these are some of the reasons that we let we were led down this path i'm just going to talk about two of these diseases in the rest of the time that i have talking about hyper IgE syndrome which have stat 3 mutations as well as another paper that was recently published by dirt jeavers and colleagues looking at stat 1 mutations with chronic mucane ecocutaneous cannidol infections so stat pathway is important it's a biochemical pathway and it's it's involved in so many things but these patients have art risk for infections and hyper IgE syndrome they have staphylococcal skin and lung infections cannidol infections and they can develop secondary aspergillus lung infections so this is the paper i just mentioned by dirt jeavers and colleagues where they looked at stat 3 and stat 1 mutations it's hard to see but these the left most groups these are the stat 1 mutation patients the middle grouping are the hyper IgE syndrome patients or stat 3 mutations and these are their controls but in general what you see just summarizing everything in this little corner at the genus level you see increased carinobacterium species in the stat 1 mutations you have decreased carinobacterium species in the stat 3 mutations you have increased gram negatives that you observe in these patient populations decreased pervitel and decreased fuzobacterialis so you we observe that there are taxonomic differences that we observe on the skin of these patients with primary immunodeficiency syndromes this group went on to do some studies of challenging pbmcs these are pbmcs from healthy volunteers what they initially did was they pre-stimulated them with either carinobacterium asinidobacter or staflococcus and then they challenged them to canada albicans or staf aureus and what this essentially shows is that if they were initially exposed to asinidobacter baomani there was a decreased tnf-alpha interferon gamma and il-22 that was produced on upon exposure with staf aureus and c-albicans in these healthy volunteer pbmcs so exposure to certain skin microbes may alter pbmc cytokine response to pathogens such as c-albicans and staf aureus this is just a snapshot of some of our work looking at patients with hybrid IgE syndrome stat3 mutations but due to the limited time i will direct you to poster 31 where julia oh we'll talk we'll walk you through her analyses that she's looked at in stat3 mutation patients but other primary immunodeficiency syndromes as well so briefly going through summarizing this part ad flares are associated with shifts in the skin bacteria we talked about the different species the specific primary immunodeficiency patients harbor distinct skin microbiota and that altering the skin microbiome may alter the pbmcs response to specific microbes but we need more studies quickly moving into the gaps needs and challenges since i'm running out of time these are knowledge gaps and then i'll go into as owen charged us with finding things that frustrate us on a day-to-day level but knowledge gaps here are evolution of the skin microbiome over life stages what are the physiological factors that contribute to the skin microbiome differences getting down to understanding skin microbiome and immunity interactions trying to expand on what has been done now but to explore that to a greater degree and fully understand how our immune system interacts with our skin microbes and again the importance of human and animal models we've talked about this but moving from correlation to causation but also as our recent survey has highlighted the magnitude of interaction between fungi and bacteria and the role in health and disease but one of the bigger challenges that i i don't think i've seen anything um been able to overcome this one challenge is skin meninginomics and that is due to the low biomass on skin so uh i do have a caveat these are my views i do not represent the views of the government but these are some of the frustrations that we encounter on a day-to-day basis standardization of protocols we've talked about this but particularly for skin i did a non-scientific survey of some of the people who do skin work here and we have different protocols i will admit that we don't follow the h and p protocol we have our own protocol that we've been doing and we've had to modify that from time to time so we are just as guilty of not having following a standardized protocol um the importance of phenotyping patients making sure are they really healthy or what are they normal what diseases particularly atopic dermatitis has several different phenotypes how do we define what is the phenotype we're studying and how do you main um especially you do that when you're at different multiple sites which sampling sites do you do how frequently what is the skin prep the time since antibiotics what sampling method is swabbing still the best way what are the critical metadata fields that are needed and it may change depending on the disease state you're looking at DNA extraction this is really critical um looking at especially when you have low biomass you um it's easily contaminated when you're talking about skin but then when we're looking at fungal organisms we had to modify DNA extraction but the primers pcr conditions just looking at various abstracts in the past and in manuscripts there are they're all over the place um quantitation this is a key question that i always get asked how do you know can you quantify what's there and we can't at this point and that is a major frustration i think not just for skin but in the entire microbiome field more microbial characterization where including genomes where we had to gene you know sequence our own malassezia in order to figure out speciation metagenomics analytical tools particularly if you have low biomass and one gripe about data submission um we even when we're submitting a manuscript and having to do db gap and sra that is a major frustration again my own opinions so then if i can move on to the acknowledgments if they will it's not there we go this um i'd like to acknowledge my close collaborator julissa gray and i have underlined here as well as photos of the postdocs work who i've highlighted here but there are many collaborators um within my own group but then also um collaborators among across different institutions but especially our patients and volunteers thank you very much for your time uh thank you so much for that great talk so we do have time for a question if there is one out there oh come on go ahead that was a lovely talk in your discussion about the areas we need to work on methods i think today as i was talking with a colleague we've seen very few slides actually on the methods that are used in any of the studies particularly in humans whether it was a wet a wet cotton swab whether it was a brush you know i think that as part of the methodological discovery process we all need to disclose or discuss exactly what the methods are as we're going forward even in presenting data such as we have here i know from the papillomavirus field we went through decades of methodological considerations that were critical so i applaud you're you know pointing these out and i encourage everybody to start including more specifics on exactly how they've collected the specimens process stored etc so we can understand that as we're interpreting the data thank you for your comments thanks for a nice talk uh when you show the results uh you show the fungi changes and you went very fast basically you didn't do much comments but i think i saw malassezia globosa increasing in the skin of the in the flare can you comment on on that uh if we're are you talking about the atopic dermatitis one this one or were you talking what or were you talking about the that one yeah so this one i will comment this particular individual was um tanner stage four and so what we see is um it's possibly related to although this was a pediatric patient that they had already transitioned through puberty and had more oils on their skin and so that's why we see that malassezia globosa is dominating potentially in this individual or it may be something unique to this individual and we just with a small and it's hard for us to tease that out thank you for that question thank you thank you okay so for our next talk we'll be uh oh sorry yeah you can clap our next talk we'll be moving on to the lung microbiome our next speaker is Gary Huffnagle he's from the University of Michigan and the title of his talk is the lung microbiome challenging old paradigms about microbes and the host respiratory tract