 I'd like to start off by thanking the leader and the organizing committee for this invitation to speak. The goal of my presentation today is both included in my title, which is to bring the audience here up to speed on this rapidly changing field of the study of the microbial communities, the microbes themselves, that we're finding in lungs both during health and disease, but then also identify the gaps, challenges and needs. And so to start with, I'd like to take you a little bit back in history because I think this puts it in the context. And so this summer actually pushes, I was just thinking about this is the 10-year anniversary of when my postdoc, Mari Nova, in my lab was finishing studies that in which we were able to demonstrate using mice that if you treated mice with an oral, poorly absorbed antibiotic, you could change immune responses in the lungs, a distal site. And the importance of that set of observations is because at that point in this idea, the study of the hygiene hypothesis, which by the way had its origins in the study of the etiology of allergic disease, one of the caveats is, let's see if I got a pointer here, had something to do with antibiotic use, so high antibiotic use, low antibiotic use, and this is coming on about 15 years or so of epidemiologic data that pointed stronger and stronger and stronger that antibiotic use correlated with the development of allergic disease, but not a single study that could actually demonstrate that that could be the case. And so that's what that was. Interestingly the results as I presented them were well received at the meetings I was at and we were actually asked to write a review on the topic, a review on a topic that never existed, that was actually quite an interesting challenge to us. So we kind of made it more of an opinion piece back then of like okay here's where the field stands, here's the epidemiology, what are the possible mechanisms that could drive this? Since those studies, there have been now a number of studies that have come out that are all addressing the same issues and have shown the same sort of thing that whether you use poorly absorbed antibiotics or you use germ-free mice that if you disturb or alter the gut microbiome, the microbial interactions in the GI tract, you can change immune responses in the lungs, not just allergic which is on this slide, but also immune responses to viruses. So the question was then how does this work? And so just a little in our interesting side note, so as we put these studies out for publication, nature turned their response was these are not broadly applicable, go away, so that was a 39-hour rejection, science did not send us out even for editorial review and later on as we went through review processes, this is the question that keeps coming up again and again, how does it work? Okay and I love this slide actually from Jacques and Larry's studies and so Jacques showed the other day or yesterday about the bouncing ball along trying to get from one point to another and this is basically how this field has moved for the past decade which is as we try to go from one point here to trying to get to the question how do things work, we've run into many obstacles and I say we, it's not just us, it's many other laboratories as we bounce around and what are those obstacles? Well the gap is trying to understand it and the obstacle is the type of science we need to do when we're talking about distal site communication and that's this idea that this is a complex in vivo system and how do you get this by study sections in which they're dominated by reductionist biologists and so I know I'm preaching to the choir here because we deal with interdisciplinary research all the time and things like that but this has been a huge, huge challenge to this field and so but we're not without ideas of how this works and so the idea is that so as you inhale antigen we know a lot about how immune response develop in the lungs and their outcome of that pulmonary challenge but studies showed many years ago that sampling itself it's hard to deliver something directly to the lungs whether it's intranasally, inhalation, et cetera things get swept up and swallowed and they go to the GI tract where the immune system in the GI tract picks it up and now as a number of studies have been highlighted so far in this meeting about the potential of immune regulation in the GI tract affecting sites whether it be the lungs or the brain or whatever the idea you can move around and then of course the idea that the microbiota might actually influence how immune response is developed in the gut we know that or you know a lot of studies here are talking about this for local responses but then the potential that this could affect this immune regulation this way but now there's more evidence coming out that microbial metabolites circulate and so it opens the possibility that metabolites might also be affecting this response here and there's actually a wonderful set of studies by Ben Marsland in Switzerland it was on that I've seen at meetings so far where he's looking at short chain fatty acids and their ability to affect pulmonary immune responses when delivered systemically but one of the sets of reviews that came along during this whole process was well what you know are your antibiotics affecting the microbes in the lungs okay well besides the fact that these are poorly absorbed antibiotics and later we've shown that it actually would not have any effect even on an infection what a stupidly absurd question okay 10 years ago because look at a textbook the normal lung is free from bacteria okay and so in fact is that one of the drivers that when you look at the human microbiome project there's nothing there in the lungs there was no sampling of the lungs in the original surveys and so the question about paradigm shifts is what I'm trying to focus on here and so a few years ago then a landmark paper came out from Marcus Hilty and Bill Cookson over in England that actually showed that in asthmatic airways well first of they showed that this is using a clone libraries that in allergic disease A there was you could isolate microbial signatures from the lungs of healthy individuals they changed during asthma there was also some COPD data in there and so this is pretty fascinating and so since that point in time there have been a number of disease in which the lung microbiome has been reported to be altered I put cystic fibrosis on here because actually that disease in and of itself has a really long history of having microbial colonization of the airways and it kind of it's cheating it doesn't really count because we know that with all that mucus there really changes the architecture and you've been able to culture a lot of things out but for a lot of other diseases culture-based processes have been fell short but now we know in asthma COPD and in bronchiolitis or Blitterans syndrome that follows lung transplantation that we can find altered microbial communities so let me tell you a little bit then about airway anatomy because this is very important when we talk about where are the microbes or where could they be and so need to realize that that you that for anything that's coming down here this tube it starts in the mouth and the nasopharynx so it's a fairly significant microbial load up in the mouth and nose and then it takes a 90 degree turn okay and then you head down the throat you get a split whether you go down the esophagus or into the lungs and then the lungs begin to branch and branch and branch at the very end of those branches are the alveoli and so there is one little area here so where the larynx is I mean potentially can serve as kind of a dam between the upper and lower airways we know that they're cilia cilia epithelium that are pushing the mucus up and there's lots of turns and branches and in fact it kind of this is what the lung looks like so you get this tree and you get things that are subject to gravity something areas that are not and actually if you look at a disease lung we later on published a study looking at x plant lungs these are lungs are pulled out of individuals have advanced COPD pull the tissue sterile look at different regions and you can actually see regional heterogeneity in disease of actually the microbes that are growing there so what are the potential sources of microbes in the lungs they include nasopharyngeal aspiration inhalation the air is full of bacteria reflux and aspiration and then probably only if you're if there's a problem would you actually get microbes coming into the lungs via the bloodstream so can lungs be considered sterile and if so what do we mean by sterile in other words is or does it mean that they lack microbial exposure well no way I mean they're at the end of the they're the cul-de-sac at the end of a very busy street of the microbes we find is or actually microbial metabolism going on are they alive are they metabolizing that's a question that we're is in active area pursuit in this field what about replication we're finding microbes there are they actually replicating so if they're alive or they just persisting again an unanswered question and what about colonizers like in health certainly in disease we can find them but but at what point in a transition from health to disease do we actually now get colonization so it really raises a question if there's mic if the lungs the healthy lungs are exposed all the time to microbes and microbial signatures and there are live microbes going down there what do we call this constant this persistent low-grade flow of microbial immigrants into the lungs what do we call this microbial flux the microbiome of the lungs so now how do you sample the airways this is another issue in this field so first off you can you can get sputum which is basically a mucous plug in a sense it's mucous from the sort of upper airways it can come from a few branches down but you hack it up okay and it can be spontaneous or induced so spontaneous is usually if you're sick otherwise squirt some hypertonic saline in the back of your throat and you hack it up but then there's bronchovial lavage so sticking a tube down there to rinse out the airways but in that as you stick a bronchoscope down there you could put a protected brush there's a little cap at the a little waxy sort of plug the pops out as you get down there and you can brush the the epithelium you could do a biopsy either through the bronchoscope or you could come potentially through the the chest cavity and then there's the concept of sterotitial sampling which largely is only going to probably occur in either cadaverous lungs or ones removed for lung transplant so the gap is determining the degree of bacterial transients versus persistence versus colonization in the lower airways and the challenges are the types of sampling to study the lung everything is invasive okay so so the rules are just different right from the get go of how we can handle samples we can't do longitudinal sampling I mean when you stick a bronchoscope down there with the exception of lung transplants where you do surveillance bronchoscopies you you you can't really go more than two or three samplings in an individual over a period of a couple years even and then of course as mentioned the bronchoscopes go through the nose they go through the mouth there's a potential for contamination samples from nasal or oral microbiota and so a couple to deal with that last one a couple bioinformatic options have been put forward which are very good so one from Rick Bushman's lab on using a single-sided outlier test and another one that Tom Schmidt in our group is actively using and it's been published in the lung HIV microbiome project which is called the neutral community model which is I mean the idea is that you're at the end of a flow okay so how can you tell the difference between a scope going down or just what naturally flows down and then is there a selective pressure in that site but you want to know what's down there well this gives you kind of an idea and so if we do rinse of the bronchoscope before it goes down we get using 16S PCR QPCR in a five mil bronch sample it's about a thousand copies in a healthy non-smoker it's about tenfold higher than what we see over baseline so maybe about ten to the fourth copies per five mils of BAL if you have disease so interstitial pulmonary fibrosis or lung transplant you can see the numbers go up so give you an idea what do these communities look like compared to like the mouth and so this is from a study in our group in which you looked at a left BAL right BAL and then other and then the oral wash and so they're color coded so now here's the blue which is the left BAL and so is the centroid of all the BAL samples from our subjects here's the black this is the right BAL you can see it doesn't matter if you're left or right that that the centroids are smack on top of each other in terms of what the populations look like but here's the mouth and the centroids are different you can run statistics on it and this as a population as a as a collection is is significantly different than what you see so the mouth is significantly different than the lavage however we talked about individual to individual variation and so what we can also do is go back to these samples and measure a distance metric so break Curtis theta yc more C to horn you name it but this case that we did a break Curtis distance between samples so the oral wash of a single individual versus their left BAL or the oral wash versus the right BAL and what you see is a spread so you see some individuals in which rinse of their mouth and what we get out of the BAL is wickedly different and you get some that are very similar what does that mean we don't know but if we run some of these tests like for example the single-sided outlier test with it here's one of the low break Curtis dissimilarity so so therefore they're very similar and we go up here to the outlier test and you can see most of what we find this is oral versus lung is being found in both and so here's another example okay everything's falling along the line but what about a sample up here you can see that what we find in the oral what we find in the lung are very different there's another example of that okay so we so we don't know what it means when a healthy individual comes in and their bronchovilla lavage looks like an oral rinse or if it doesn't look like an oral rinse and again we can't follow over time what that actually means so what I think the field so far the investigators are involved because there's obviously as you can imagine there's some debate about sampling about how to handle contamination things like that but I think what we can all agree on this point is that when healthy the microbial load in the lungs is low and the BAL samples contain a predominance of bacteria taxa that's also found in the mouth we know the bacterial diversity in the lungs is very low actually we can use that to our advantage because we can do pyro sequencing and we don't get that many types of OTUs and we can actually take consensus sequences from those OTUs from the 16S RNA gene amplicons blast them backwards and really come up with only one genus that it could be and sometimes we can get as low as a species because there's no other options around it and so it's actually very useful that way so we again and we know when some individuals are some differences suggesting it's selective pressures can exist in the lungs for elimination persistence colonization and growth now if we move to disease so here's our healthy individuals in red here's individuals that had a lung transplant and these are bronchovilavages and this light green hopefully you can all see it is interstitial pulmonary fibrosis and you can see that if we call this sign the normal cluster you can see there's some lung transplants that are up in the normals there's some IPFs up in the normals if we focus on interstitial pulmonary fibrosis for just a second so we'll call this the IPF the healthy group and this is the the other group that doesn't look healthy and we actually ask who are the bugs that are there who are the bacteria that are there what you can see is in healthy individuals we get the signature that not only we get but they get it in Europe they get it in labs all around the country which is the dominant organisms that are coming out of a bronchovilavage are prevotelavinella and streptococcus and then quite often things like fusobacteria and nyseria and so when we look at our quote-unquote normal or IPF one group we get the same sort of organization but if we look at this other group suddenly we get pseudomonas we get escherichia so we're suddenly getting some gamma protea bacteria some gram negatives that are coming in there now interestingly if we go look at the lung transplant recipients our healthy controls we actually found surprisingly an OTU that when we blasted it came back as pseudomonas fluorescence and interestingly it was in our only in the lung transplant we have never found it in our healthy controls and what it act we find it only when in our lung transplants when the number when basically the relative ratio of prevotella reads is low so in other words when it stops looking kind of like that mouthy lungy sort of pattern suddenly we get pseudomonas fluorescence there in a large number of individuals and so pseudomonas fluorescence should perk the ears of a few people in the audience because like Balfour and Jonathan Braun and anyone else who works with Crohn's disease because you make antibodies to it at a very high rate if you have Crohn's disease yet it's not a pathogenic organism or at least it's not believed to be so why is it there why is it in these lungs and so to get to that question so let me summarize this point and say that when disease the microbial load in the lungs increase in BAL samples now often contain numerous bacterial taxa they're not found in the mouth indicating that there are selective pressures in diseased lungs so the challenge now to my question I just ask you is that studies only involving human subjects will never demonstrate causality no matter how large the cohort and so in vivo animal studies model organisms and vitro experiments are needed to delineate the mechanisms they have to work hand in hand you gotta go back so so translational research is iterative you go back and forth back and forth back and forth and so we need support for animal models in the study of the human microbiome otherwise how do we get back to this whole thing about the hygiene hypothesis and why these elements are on the different tip part of the balance so to address the question why is pseudomonas fluorescence there in the lungs we've been working for a long time on a model in which we can generate allergic or airway inflammation by multiple exposures to aspergill's fumigatus spores and so this shows you after four challenges eight challenges you get lots of leukocytes in the lungs lots of inflammation th1 th2 th17 the lungs are kind of really messed up well what we did is we went back and and and look at those animals and say wonder what happened to the lung microbiome of those mice and if you go over here this is again pyro sequencing look we says bloom of gamma proteobacteria that happened after four and eight challenges this chronic inflammation that's going on the lungs and if we look pull the otus out the one that jumps out the pseudomonas fluorescence oh granted we got lucky okay because we didn't that wasn't the point at this point we've actually gone in and taken the inflamed lungs and put pseudomonas fluorescence in it and it does indeed like inflamed environments and so it raises a question now gamma proteobacteria so pseudomonas fluorescence is not pathogenic but it's a gamma proteobacteria escherichia is a gamma proteobacteria gram negatives okay it's a common theme that we've actually seen also in the gut that inflamed sites favor the growth of gram negative organisms so the final case that i want to show you here to show you how our paradigms are changing and our thought process is changing is we've known we've assumed for a long time that klebsia and pneumonia is an etiologic agent of pneumonia however as studies from our lab and my collaborators we can take two different strains of klebsia and pneumonia that were isolated from patients of gram negative pneumonia that responded to antibiotics etc put them into mice one will kill the mice and at the same dose the other one they'll walk away i'm sorry other way around so one will kill the mice one want to walk away yet they came from human beings that had disease that responded so what is going on there so an interesting thing popped out of our studies of the lung transplant recipients if we actually looked so this is a single individual two bales over six months in time at this point they had a they had a pneumonia by CT and they had all the other clinical symptoms they were put on a ciprofloxacin they recovered they did much better the culture was negative the whole way along the way but when we looked at it if this is a the from the pyro sequencing you can see the sort of the normal cluster in here and and when they first came in they were way different and by the way these individuals all have pseudomonas originosa up here and but after ciprofloxacin they had a microbiome of their lungs it looked kind of normal so what did that look like escherichia just dominated by escherichia but it didn't grow out okay so we talk about unculturable bacteria or difficult in the gi track and there there are things that you look at me say i don't recognize that name you know and so but what about the ones whose names we do recognize that suddenly i'm going to argue in the airways there's something different about microbial growth in the lungs that they are not able to be cultured under standard uh microbiological process uh techniques that we use now which means that the gap is that we need to understand the implication of cultural and non-culturable states of bacteria in the lungs and also i've been talking about bugs where are they okay so there's some work that's coming out and i'll build cooks and things like that i mean and so who's live who's dead where are they but i just gave you an example of somebody who clinically responded to the fact that they've got a bug in their lung but we couldn't grow it out and we've actually now we've got one set of pilot experiments where we've done in the sense the same thing we've taken a not a cultural bug put it in the an inflamed lung and watch it become unculturable um i don't know what it means though so the bottom line is we need new cultivation strategies when we start to study the airways so the overall challenge of the field you know i can't underline this enough okay we need more consistent supportive peer review of microbiome lung proposals granted it's all aimed at my world but um but but my point is is that whether it's looking at the microbiome that's in the lung or the role that distal communication so i guarantee that anyone who's trying to do microbiome nervous system or or gut microbiome nervous system or gut microbiome some other site other than locally um your proposals are going to be open for field day because there's so many things that can be poked holes at and it's hard for the field to move forward we also have to accept that sampling of the lower respiratory tract in humans will be imperfect i i cannot even think of how we're going to solve that problem okay because of ethical reasons um and so we're going to need to utilize animal models to close this gap and so this is the research group that i showed some of their work both in terms of in my laboratory but and also my wonderful group of collaborators at u of m and so i thank you very much for your time well thanks gary for that great talk and for so clearly articulating some of the gaps and challenges in this area uh so we do have time for a couple questions we have over there so um just in the spirit of a little bit of dialogue about this i i none of us want to interfere with the exquisite NIH review process but um and i meant that sincere sincerely but uh the uh i i wonder if one of the things that we could talk about is the possibility of sort of a trans institute uh review panel that was microbiome friendly if i just i would just introduce that idea and i i've spoken awful lot of people that have related issues in terms of just you know are there panels out there that are sensitive to so i'll throw my opinion out there which is that my first thought is that i like that idea but then i realized it's a double-edged sword okay and so like so i sit on study section so why we're not supposed to talk about the f word when we review a grant okay um you know the bottom line is is that you know that when you sit on study section if there's a pile of a hundred grants that come in and and you know that the institutes are are funding it around 10 percent ish you know that 90 of those grants are going to go home and tell you to stay here so now what if we take everybody in this room and take all of your grants and put them into one study section that means that 90 of you are going home so there is a danger by lumping everyone together as opposed to moving across because if if we at least keep things spread then you're looking within an area within a discipline what's in in a sense what's the relative importance of microbiome research versus a cell biology versus an epidemic that sort of thing but the problem is and my big beef with a peer review system is since when is peers three okay you have a study section of 20 something people and that very first pass to get through you have three people read and then that's it that can determine a fate so basically it's like the un security council one individual can trash something that part needs to be adjusted a little bit when you're dealing with interdisciplinary or risky research or discovery okay because when it's not hypothesis testing and it's hypothesis generating boy they it gets really risky but I do think an active conversation needs to be made needs to be put up that's probably a good topic for our later discussion too we'll take one more question while our next speaker comes up so regarding your non-culturable escherichias since they came from a pneumonia which there may have been a biofilm so in the biofilm field people talk about persisters you think it could be something like that yes I do actually I think I definitely think that that's and so there's two things at play here one is the idea of persisters and any other idea is that you know the microbial load even in pneumonia you know even this thing is is not necessarily high and so what we may end up having is sort of in terms of ecological sense terms like islands and you know or whatever oasis is in deserts okay so within the lungs but I do think that those are mechanisms those viable but non-culturable mechanisms are are going to be at play here thank you our next speaker is Dr. Vincent Young at University of Michigan he's going to enlighten us with a talk entitled the microbiome in infectious and non-infectious gut inflammation