Okay, thank you very much for the invitation. What I'm gonna do is give a little bit of the basic grounds of my microbiome work and some view on what my meaning in the setting of ARDS, but really we have the other two speakers, especially Bob, that has a lot of much more experience in ARDS, so I'm gonna kick off with the basics. These are my disclosures, mainly grants that fund the lab to do the work that we do, and we're gonna talk about microbiome at this stage and age. People know what they mean, but basically, the thing that I want to highlight is that these microbes inhabited us, they're at extreme high number. They have, we have more microbial cells than human cells, and if you think about genomes and genes and functions contained within these genes, the sum of microbial genes actually carry many more functions than our human genome, so there is great potential of what they can be doing. We haven't paid attention because basically our understanding of the microbial world has been focused for many years in trying to isolate these microbes, but we know that microbes do not live in isolation. They live in complex communities, and now we have techniques that allows us to examine this, and we do it basically by recognizing and measuring microbial products, which could be DNA, RNA, protein, or metabolites, and that allows us to, in this culture-independent methods, to comprehensively characterize microbial communities. Now, when I study medicine, the lungs were sterile in textbooks, and using these techniques, we know that now we can identify microbes that have adapted to live in extreme conditions, and these are two examples. Now, in the hot springs, in Yellowstone, or in Antarctica, extreme, horrible conditions, microbes can adapt to it, so why wouldn't be able to live, or to seed, or to be present at times into the lower airways, where there's moist, there's a nice temperature, and there's a nice pH? So, in the last several years, now using these techniques, we can uncover the role of these microbes, and we understand that microbes affect our immune tone, and are being affected by our immune tone, and it affects how we interact with pathogens, and this can be playing a big role into areas where there's a lot of microbes, such as the oral cavity and the gut, and through indirect movement of microbes and movement of inflammatory cells, they can affect the lung in health and disease lungs, and this is, when you think about the gut, people talk about the gut-lung axis, and there's excellent investigations in this area, and some of that might be highlighted a bit later. My lab has focused on the fact that microbes also enter the lower airways through the upper airways, and the sole presence of microbes in the lower airways, even if it is transient, impact the host immune tone in our lower airways, and imprint how our immune system will respond. So that happens in health, but things change as disease occurs, and ARDS, which is the topic that I was asked to talk about, imposes quite a unique challenge and unique situation. If you think about it, these are patients that have an invaded airway, they have a connected tube, the mechanism for coughing is impaired, so there's biofilms in the tracheal tube and in the lower airways that will affect the dynamic of immigration of microbes, and the dynamics of emigration of microbes. There's also factors, either host factors or environmental factors that will impact the ability of microbes to see the lower airways, to reproduce, and to interact with the host, and there are some examples here that are related to ARDS, that are important, that were mentioned in this review, that's why I thought that it was pertinent to put here. But if you just think about oxygen, I'm sure that Bob was gonna give some examples of this, people are exposed to different oxygen concentrations that will impact the ability of microbes to reproduce. So if you are a facultative aneuro, or an obligatory aneuro, you will behave differently than if you are an aero. So these are important considerations that makes the system complex, and we need to understand. Now, if you think about microbial work, or microbial work in the setting of ARDS, there are a couple of things, there's one slide that is missing that highlights some of the work done here in UCSF, showing that if you study patients, for example, with blunt trauma before, at the time of intubation, you can identify microbial signatures that are associated with the later development of ARDS. So in a way, the microbiome might be predictive, or might be associated with that development. If you study patients with established ARDS, I like this figure, because it highlights that things are not simple. And this is a co-occurrence networks of multiple different microbes that you can identify using these culture techniques, culture-independent techniques, when you study the lower airways of patients with ARDS due to SARS-CoV-2. So we did this by obtaining a lot of samples from bronchoscopy on these patients, and the co-occurrence network shows how these microbes are associated. So when you have one of them, if you see another one that is close by in this network, it means that they tend to go together. And the color on the nodes represents red if they're associated with worse outcome. So if you look there in the center, you have SARS-CoV-2 that is red, and not too much surprise, it's red because the higher the relative abundance of SARS-CoV-2 in the lower airways, the more likelihood you're gonna have a worse outcome in this cohort. But you see that it's not just SARS-CoV-2 that is red. There are many other microbes present in the lower airways that might be co-occurring with SARS-CoV-2 that are also associated with poor outcome. And most of it might not be even a virus, they are bacterial or fungi. So let me expand on that. If you take patients with ARDS and SARS-CoV-2, and you now measure bacterial load by using just a ddPCR measuring quantity of total 16S rDNA, which is a marker for bacteria, you see that patients that die have higher bacterial load than patients that survived. And when you look at the identity of microbes in those lower airways, we can establish this analysis where if there is a circle in color, means that there are, to the right, it means that they're associated with worse outcome. And the size of the bubble, it's related to how much of those microbes are, what's the relative abundance. And you see many, many bubbles that are in color. That means there are many taxa other than SARS-CoV-2, just on the bacterial part of the microbiome that are associated with worse outcome. And if you were able to read all the taxa names there, the ones that were significantly associated with worse outcome, they're not the usual respiratory pathogens that we all suspected at the beginning of the pandemic. That's why we stopped treating patients with antibiotics and the microlabs start saying, don't send any more cultures in these patients, they're all negative. Well, but there are stuff that were associated with worse outcome. And if you look at that bubble, that big bubble that you see there, which represents a high relative abundance of one micro that was associated with worse outcome, it ended up being mycoplasma salivarum, which is an oral commensal. So there might be a role of these type of oral commensals that through emigration in the lower airways might be contributing to the inflammatory process or setting the inflammatory response in the setting of SARS-CoV-2 infection and in the setting of ARDS. This data does not come in isolation. If I show you this paper from the Blue Journal that came out a little bit later from the Netherlands, show something similar. If you measure bacterial load or you measure fungal load, you see that patients that have higher levels, higher amount of bacterial fungi tend to have worse outcome. Bob did an investigation before the pandemic where he was measuring the bacterial load in patients with ARDS. He stratified the patients based on how much bacteria was found in those lower airways. And you see that the group that have higher bacterial burden, they tend to have worse outcome. And when you start looking at those bacteria, you start seeing, again, you see not necessarily the classical respiratory pathogens. In this case, there's a nice story about enterobacteriaceae that I'm sure you're gonna expand a little bit on it in a bit. So these type of techniques are quite powerful now because we can use it to develop and identify microbial signals that are associated with worse outcome that are beyond the pathogen that we've been looking for for years. But you can also compound this with other piece of information from this type of culture-independent techniques. You can look at host signatures in the transcriptome or in the cytokine measurements and develop host signatures. And we did this in the setting of SARS-CoV-2 where you can combine the two signatures, the microbial signatures and the host signatures to develop models that are highly predictive of poor outcome. This is important, not just because it might be of some diagnostic utility, but also because it may uncover another mechanism by which the microbiome and the host immune response may be affecting clinical outcome in these patients. But this idea did not come on isolation. This was a stolen idea from this paper, from a group that here in San Francisco where they actually were studying patients with lower respiratory tract infection comparing to those that do not have lower respiratory tract infection and using similar approaches as I've been describing. No metagenome to look at microbial DNA, metatranscriptome to look at microbial RNA and host transcriptome to look for host signatures. They can identify, use these techniques to identify more pathogens that you would otherwise, but you also can identify gene signatures that are associated with, in this case, lower respiratory tract infection diagnosis. And you can use this to, as I was showing, to develop a model of microbial prediction, a model of host prediction and a combination of the two that can start dissecting possible mechanism by which microbial host interaction are playing a pathogenic role in this condition. So if you think about this general structure of ARDS, it's clearly that the lower airways environment changes tremendously. Now you pass from not having much in the alveoli to now have a lot of debris, a lot of proteins, a lot of degranulation of neutrophils and dead cells and debris. So if these are different conditions in the lower airways that are providing different carbon sources for microbes. So I'll measure how much nutrient protein exists in the setting of ARDS. And you can see that it's many folds higher than what you would see in health individuals and also higher than in other respiratory conditions. So this will affect the microbial dynamics in terms of what bacteria will grow in the lower airways. So it's not surprising that we're starting to find some signals. Bob has described gut associated signals. Fathers have described the presence of mixed oral commensals in the lower airways. And ultimately what's happening is that in the setting of ARDS, the structure and the topographical distribution of microbes starts changing. When if you think about a normal, relatively normal individual, the lung microbiome looks very different than everything else. If anything, it looks more similar to the oral cavity, a little bit to the nose, very distant from the gut. In the setting of ARDS, that difference is that topographical difference starts becoming blurry. And the real question is, can we identify these biotic signatures that are on the causal pathway that are contributing to the inflammatory injury that occurs in the setting of ARDS? I think that's the million dollar question and that's where investigation should go. But we can't do that just with human studies. We need longitudinal and we need preclinical mouse models or preclinical models. So I just want to end here. I hope I convinced you that the lungs are nostril but are rather frequently exposed to microbes and their byproducts. And this condition changed tremendously in the setting of ARDS. And those are things to consider when you think about studying the lower oral microbiome. But ultimately, if we can dissect the mechanistic links and the microbial functions involved, there might be a novel path in which we can identify a novel target that can impact inflammatory damage and the pathogen susceptibility that we'll ultimately see in these patients that are a big part of what determines the outcome of patients with ARDS. I'm gonna stop here, just acknowledge the people in the lab and the funding that supports this work. Thank you very much.

microbiome

acute respiratory distress syndrome

ARDS

immune system

lower airways