Thank you for that wonderful, undeserved introduction as well as I want to thank the society for giving me the opportunity to talk today as well as my previous speaker for setting such a good bar and a good review to sort of discuss. Today I'm going to be delving in a little more specifically into some of the immunology that she was able to review and talk about some of the human translational data we've been working on. But before the only disclosure I have is the NIH funding. So a lot of what we talk about now is post-ICU syndrome and it's important because of SCCM, because of surviving sepsis, because of what you guys do out there, our patients can actually survive the acute phase. And although there's still of course acute mortality that we have to deal with in interventions, the dynamic and the phenotype and the outcomes have changed somewhat and that instead of dealing with acute mortality, we have a lot of sepsis survivors. And when I say survivors, I mean it depends how long you think they've made it, but frequently we actually are able to get these people out of the hospital. But the question is what happens after that? And that's a lot of where I think the post-ICU syndrome stuff comes in that we've been discussing and SCCM sort of heads up. And it's important to understand that as you sort of, this is an example of sort of potential outcomes that could occur. And again, things are moving away from number one where you see early death and then you have two different basic populations long-term. Those that rapidly recover and in this definition it's those who are out of the ICU with no longer having organ failure after two weeks. Remember that as Cliff Deutchman will explain that sepsis requires and is defined by organ deficient failure in an infected patient and that's important to understand. But if you ever wonder why a lot of these cytokines and anti-cytokine and chemothyme therapies aren't working, it's because we need to keep pushing towards precision and personalized medicine. No one in this room, unlike maybe potentially an antihypertensive med, can have the exact same thing to establish homeostasis, which was already mentioned, to allow you to sort of get back up and functioning and walking. So some people you should actually leave alone. And you're not always able to actually predict this in the first 12 to 24 hours where you're on pressers and antibiotics and all the acute stuff is going on. So you got to give it a little bit of time maybe. And then there are those where you may want to focus at least more of your interventional drugs if you're looking at least from an immunologic standpoint. Of course, there are other things you want to look at, muscle, cognition, things like that. But those that enter chronic critical illness and this is just one definition. It's been defined multiple ways in the ICU, but it's the concepts sort of the same for all of them. That you are still in the ICU with some form of organ insufficiency after at least a week or two and it doesn't look like you're heading back to normal. So you're not going to be going home. You need support. You need help. At best case scenario, go to a skilled nursing facility or a SNF. So these patients are the ones that if you sort of study them long term, you'll find out that within a year or two, they haven't gotten back to normal. And quite a few of them may have actually succumbed to other illnesses or things and died within a year. Most of them have very hard times actually, not most, but a good percentage of them have a hard time doing activities of daily living. They're frail. They have catabolic issues that are associated with that. Their cognition isn't the same. Ask anyone in a sepsis survivor group how their memory is, what's going on. And so in that sort of situation, these are the ones that could potentially, with the right intervention, if we understood the complexity of what's going on, could be sort of put back on the right sort of direction. So as part of that, we decided to look at a specific cell called a myeloid-derived suppressive cell. Now what is that? This was first discovered by Gabrilevich in the cancer population. And instead of sort of having cells that differentiated to appropriate monocytes, you get exactly what you're hearing, which is a myeloid cell that is in large quantities circulating in tissue, can go to tissue circulating in your blood. And that can immunosuppress other cells. Most of the time we talk about lymphocytes, but in this case, it can be all sorts of different cells as they've shown. And the question is, well, are these playing a role? Are they similar to cancer? What's kind of going on? Because to date, it's not clear whether we can actually use the same cancer drugs. Because for instance, if you sort of wipe out all MMDSCs from a cancer patient, you can kind of treat that like a hemlock patient where they can be in a room, they can be isolated. But for sepsis, smoldering infection, other kind of stuff, removing someone's myeloid cells could be potentially catastrophic. So the question is, what's really there? What's circulating? How's it interacting with other things? So we had to start with, okay, precision medicine. What's actually there in patients that do better versus do worse? Now we have done some preliminary data. Dr. Brackovich was a part of this work where we looked at least in surgical sepsis, and we found that patients that have continued elevation of these cells, as defined by phenotype and a whole bunch of things we like to do in the lab, continue to have worse outcomes. And that includes, again, both neurologic and frailty, as well as survival and repeat infections. So we at least know they're associated with it. And when we take them out and we look at them in a Petri dish, they do meet the criteria of myeloid-derived suppression cells. They suppress T-cells. They have a different cytokine expression. They do sort of a lot of things you could read in the literature. But the question is complexity. So there's sort of a monolithic view that used to be, okay, like you learn in med school, this is this cell and this is this cell, and this is how it's defined, and a monocyte can only be this. And a classical monocyte has to have this on the outside and this one and that. And what's a little more kind of like you see in a sepsis patient is it's not so clear cut. You're not always in inflammation or immunosuppression. It's kind of a mix. And when you see this myeloid differentiation that's supposed to occur after sepsis, what ends up happening, at least in those patients we think happens, at least I'll try to convince you that happens, is that the differentiation pathways get messed up. You're not returning to homeostasis. You don't have proper effector cells. And you end up in this kind of mush that you see in B, which is the emergent view, which is this complex, I guess I could use a term to make you laugh, myeloid mushuga. Everything's kind of messed up instead of doing what you're supposed to be doing in terms to have an appropriate reaction, appropriate response. And remember, this isn't all just about infection. This affects other parts of the body as well. Myeloid cells can affect how your brain reacts and does things and also secretes things that cross the blood-brain barrier. They infiltrate tissue, including muscle tissue, and interact with their stem cells. So it can act, at least to some extent, as part of the unifying theory about why this systemic effect is going on if someone can't get back to what was their homeostasis. And just to be clear again, our homeostasis is all a little bit different. So we can't put everyone into one box, whether it's the amount of IL-6 you have or the amount of this. It has to be a little better understood, a little more complex. So basically what we have done is we took healthy controls, we took acute sepsis patients and chronic sepsis patients that met the criteria for chronic sepsis, those that met the criteria for CCI, like we talked about before, and those that met RAP. We used peripheral blood mononuclear cells because for multiple reasons you really need to pull out some of the neutrophils in order to actually analyze these cells properly. I won't get into that. Happy to answer it later. And then we did a lot of what you hear a lot about now, which is single-cell RNA-seq analysis. And it's a complex thing that, again, I'm happy to answer all the questions after the talk about how we did this analysis and that. But working with some Class A statisticians at UF, we were able to sort of do what's sort of standard UMAP analysis, clusters of genes, to see exactly what's going on between these two groups. Can we identify differences in their myeloid populations? So this is just an overview. We take all the genes, all the patients, all the cells, and they're represented by dots, the genes that are being expressed. And again, you don't have to be an expert in single-cell RNA-seq to see if you base it based on patterns, based on color. So red is four, you sort of have that maroony green, you know, for 14, the blue. You can see that they don't all line up perfectly. And that means that there clearly is transcriptic evolution of what would be considered the same cells if you looked at them on a flow cytometer. Or if you said, okay, I'm defining this cell as CD blank positive, CD blank negative. But what you can see is, well, they really don't look the same at the different time points. And then if you break it down based on some of those markers, you can also see whether it's a classic, non-classic monocyte, what are known as neutrophilic PMN MDSCs or monocyte MDSCs, as we sort of mentioned before, the different sort of types that occur. You can see also it's not sort of the same transcriptic response. So you have this overall difference after sepsis that kind of occurs. And then if you try to break it down by specific cell types, again, the delta log versus the delta P value, this is like showing you like for one gene, the change that occurs versus baseline in A, you know, if you look in the A panels over time versus their log change. And just to summarize it, again, this is an overall summary concept. You can see that red dots aren't where the blue dots and where the other sort of dots are and there isn't as much overlap as you would think, although you can see sort of the gray in the background. So again, what you're seeing is there are different cells for different people for their outcomes and they have different effects. They're functionally, when we take them out, they act differently. You can also look at the Venn diagram in B and see that, oh, yes, there is a lot of similarity, but there's also quite a bit of difference. And if you're starting to think about interventions, like she mentioned epigenetics, whether you're looking at other things to sort of go in and modify a cell and how it acts, these are sort of targets that you're going to want to approach as you go down the road. And then if you sort of look at C, that's basically overall, if you take all the genes and you ask a program, like, what could you predict would be up or down regulated? You can see, again, that it's not the same in every cell type, whether it's in the acute phase of the response or when a patient's defined themselves to be recovering or doing poorly. Again, this is very similar. Again, there's the neutrophilic type of MDSC and the monocytic MDSC. This point of the talk is not important to understand the difference, but they are different. They play different roles in different type of cancers. And again, what you can see here is that you don't have the same cell. I couldn't take the same cell phenotypically and put them up against each other and say, yes, this is what's being transcribed. This is your antigen-presenting gene. They're different. What's circulating is different. It's not the same. So again, myeloid meshuga. So instead, when you look at how, instead of sort of the classic way we think of myeloid differentiation, which you can see here is after sepsis, this concept of everything's kind of all over the place, you can do quite a bit of back and forth. And it's not quite as simple as you think. And thus, one single intervention, one basic concept, one cytokine may not be a way to get these cells to evolve or get a person to retain to homeostasis. So again, here we go. What do we have here? This is the interesting part. Again, this is a way of demonstrating all the different cells that have been present. Again, you don't have to be an expert to see the difference in colors between RAP and CCI. And of interest, there's a novel cell population that we had to come up with a name with. So we called it the hybrid MDSC. That's about half mono, half PMN. Hasn't been described before. It may be just something in the differential pathway, but we only found it in those patients that are chronically ill, again, showing that there is an absolute difference in these patients and how they respond, how their immunology is responding to sepsis long-term and not returning to baseline. This is just another way of analyzing it. And again, we're just trying to highlight the increase in early progenitors and how different things may sort of be occurring in these different subpopulations. Again, the acute phase where they're all together as opposed to those that recover versus those that have done poorly. Again, they're more similar than you might think, seeing that there are only sort of seven genes that sort of differentiate HMDSCs from the other ones. But again, statistically and using the programs, these are different cells. They're not just the standard cell you would say if I'm going to define this as an MDSC. So again, it's kind of all over the place. And then if you look at this, this is a way of representing each cell type with all its genes and all its patients put together with the thickness of the line representing how strong it is related genetically to each other. So you can almost use this as a way of determining differentiation. So distance and strength of the line means how close they are to each other. And if you start at the base, that's where your basic neutrophilic type MDSCs are until they kind of head towards the monocytic and then your terminal differentiated monocytes that you typically think of using to introduce an antigen presenting response to help kill bacteria, get B cells and T cells going, et cetera. This is another way of sort of analyzing that, showing you the direction that they potentially go. So again, you can see on the left, you start with your early cells and as it kind of goes through all the way to terminal monocytes, MDSCs, you can see that this isn't a clear simple pathway. It is not the classic pathway. You do not have simple cells that can respond to simple things. And this is using the RNA velocity volumes. And to summarize again, if you look at, you can use unspliced mRNA from transcriptomics to say, okay, this is more likely to differentiate. So you can see progenitor MDSCs, early MDSCs more likely as you move kind of down the ladder to dendritic cells, which are somewhat more terminally differentiated. And that follows kind of, again, what we've shown you in the patterns about what sort of down the pathway, what's kind of going on. Now, this is where it gets also a little interesting. There are sort of some clinical, in the cancer literature, there are some cells such as induced nitric oxide or arginase one, which are in the cancer literature, legitimately key ways of inducing immunosuppression. We keep looking for it. And we will keep looking for it because that's how it's defined in literature, but we can't find it. So these cells, when we take them out, do immunosuppress T cells, but not through the exact same mechanism. Some of them are the same. So you see the S100A proteins there. That is definitely a way that the cancer MDSCs induce suppression in certain cells and can have systemic effects. But some of the other mechanisms aren't the same, which means that if you were to use the exact same drugs that affect those things, when you try to translate already approved FDA drugs in cancer to affect this, they may not work. Which is why we have to have this better understanding of the complexity before we just start applying different drugs to sort of make this work for precision medicine. And again, it's going to take precision medicine to get people better from sepsis. This is just another way of showing you sort of, there's a little bit of yellow there, but in general, if you compare it to the other stuff, it should be, it's not upregulated in these cells the way it should be. And certainly in chronic critical illness and RAP, you're not seeing the same distribution of the S100 proteins, et cetera. So I need to sort of finish up. So again, MDSCs populations differ between chronic critical illness and rapid recovery. There's an emergent view that's been published that we seem to find in sepsis that shows that differentiation is way more complex than we thought. And there may even be interval cell types that haven't even been identified yet that are specific to certain outcomes. MDSC and sepsis have different genetic expression than those in cancer, not completely, but just surprisingly, some of the major mechanisms may not be the same and we have to sort of figure out where it's working from. And other immune responses are likely to also be similarly unique and complex. So again, I'm presenting the data, but this is the team that gets it all together to make it work. And I'm going to take it over to Dr. Brackenridge and thank everyone for the opportunity to speak today. So.

post-ICU syndrome

sepsis survivors

myeloid-derived suppressor cells

precision medicine

single-cell RNA sequencing