Hello, my name is Chris Haldanski and I have the pleasure to present my work at the 51st Critical Care Congress of the Society of Critical Care Medicine. I'm an assistant professor in the Department of Anesthesiology and Critical Care at University of Pennsylvania. I'm also charged with the duties of providing quality and improvement at the Penn E-Learn, which encompasses the whole University of Pennsylvania health system. I also work as a senior fellow at the Leonardo Davis Institute for the Health of Economics. Today my presentation will focus about the epigenetic mechanism, which will determine the recovery of the immunostasis after any critical care insult. This is my passion, this is my work, and I'm very, very excited to share some of it with all my audience. So let's start from the disclosures. I was awarded a K23 award by NIH, also received the money from the Penn Center for Alzheimer's disease. I also was very lucky to get a vision grant from the Society of Critical Care Medicine. Both of those grants, all of them, allowed me to actually present this work. On the other side, I'm founder of the Cuban Diagnostic and I'm also president of the Society of Healthcare Innovation. Neither of this association bears on my presentation. So let's talk what we're going to talk about today. So we will talk today about how the immune system performs under the critical care illness. We specifically will talk about the activation and deactivation after the resolution of the critical care illness. We're going to talk quite a lot how this process of activation and deactivation is regulated. And I will focus on the epigenetic abnormalities related to the activation and deactivation of the immune system. We will focus on the abnormalities. So we're not only going to talk how immune system can perform under optimal condition, but we will talk how the epigenetic regulation can affect immune system so it will perform unfavorable for recovery of the patients. And finally, I hope to leave you with some ideas how we can implement all this knowledge in treating our patients. So, let's talk, what does it mean, meaning of the recovery for the patients in the ICU. We all have a conversation with the families and depends what's going on. The recovery has can have a very, very, very different meaning. For example, this is the patients who probably will never leave the hospital, ability of that patient's homeostasis to cope with the stress has been exceeded, and the chances of the meaningful recovery are almost non-existent. As probably I and my audience realize, this patient will never leave the hospital and will die over there, even though his life is supported by all this machinery. There's also this recover, this is the patient who suffer very, very severe critical care illness. It's trying to recover, but recovery requires a lot of PT, OT, functional restoration, and pretty much a lot of work, and quite often that recovery does not end in the place which looks like pre-critical care illness. The best recovery is this patient. I particularly like this lady because this is the perfect recovery. This is the patient who is trying to sign out out of my hospital, gains medical advice, and that's smart. And if we think about it, please look at this patient. This is the patient who knows where the door are, so she's alerted or in the time's free. She can consent to leave the hospital, so again, she has a full cognitive faculties, and then she's strong enough to push her wheelchair towards the door. This is the recovery. It's also the recovery that we would hope that we will provide to all our patients. So what determined that recovery? I spent a lot of my work focused on the immune system, and traditionally, the way we perceive how the immune system is activated or almost any critical illness is that the immune system gets activated, then regulatory mechanism deactivate the system, and then we pretty much end up in the pre-health status, which is almost the same as before, and we call this recovery. However, how this process is critical on the immune system performance. And I say critical, let's ask ourselves, what does it mean? Are there any other outcomes which are possible of this, except this perfect trajectory? And there are. If the immune system very early gets over-activated, the patients will die from the immune system over-activation. If the immune system responds poorly, then the patients will die because the pathogen will overwhelm the patient's homeostasis, and the demise will ensue. And then finally, what we start appreciating more and more, there's not only one recovery. Some of our patients have a persistent inflammation, that means they are smoldering inflammation, and that's a phenomenon when the patients have ongoing immune process, which does not return to the pre-insult state. Alternatively, the patients may have persistent immunocompetence, and immune system of that patient is unable to perform to the standard as before a critical care illness, running the patients susceptible to the opportunistic infection. So when we think about all those trajectories, and we're talking about the immune system as the culprit, what is the culprit? And what I would like you to focus is, is to look at this pathological trajectory and realize the cure, they bent it. So there are absolutely critical checkpoint in immune system response that have to be followed and they have to be tightly regulated. Without this regulation, what will happen is immune system will fail. So a lot of my work focus on the checkpoint of the immune system regulation, and those regulation can be achieved via several ways. Those ways are parallel, redundant, and multiplexed, and what it means is that though I started one of the small part of the immune system regulation, that's not only part of the immune system, which may be the most important, it's just most important for my work. And when we talk about the regulation, what we want to focus are, for example, B-cells and immunoglobulins. The significant part of the immune system is controlled by the T-cells. But what my work on this work that I'm going to present focus on are the monocytes and dendritic cells performance. And in the next couple of slides, I will try to explain why I'm so passionate about this particular substance of the regulators of the immune system. So let's go back to the monocytes and dendritic cells and ask ourselves why those cells are so perfect, why I'm focusing on them. So if you remember Biology 101 or USMN Step 1, we know that monocytes can evolve into several cells. Monocytes can become activated monocytes, it can become dendritic cells, it can become macrophages, it can become osteoclasts, it can become a lot of different cells. All those cells have a different function, but what makes the system so precious for the immune system regulation, each of the cells has a different function and different triggers. And under perfect condition, what we have, we have a perfect functional balance of the macrophages and activated monocytes, which are the responders. And then we have adequate regulatory counterparts, which are naive monocytes and dendritic cells. When I say the balance, it doesn't mean that we have the same amount of cells, but the composition of the monocytes and its offspring is such that actually it balances both responding and regulatory function together. So where is balance, there's also imbalance. And when you're talking about the performance of the immune system over time, well, let's see what can happen when the monocytes, dendritic cells and macrophages do not perform as they should. And we do know from numerous papers published by various team researchers in the field of the sepsis, that you may have tons of the macrophages and you may not have dendritic cells. And what it creates is this condition when you have a lot of responders, but you do not have dendritic cells to control them. That will result in the persistent inflammation. Also, what I would like you to appreciate is that at the same time, the patient can be immuno-incompetent because dendritic cells are the key component which tune an immune system response to end offenders. So any disbalance results in the condition when you do not have an adequate immune system response, aka immuno-incompetence. But at the same time, you may have a persistent inflammation, which is being fewer even in the absence of the initial trigger. So one of the questions that I would ask again is how this balance or this imbalance actually can be sustained. So somebody had a sepsis, surgery, trauma, TBI, you name it, and you test them several months later, and they still have this monocyte-to-microviruses disturbance. If that would happen, what would fuel that process, even though the initial culprit disappear? And one of the things that we can talk about right now is something called epigenetic. And epigenetic is a branch of the biology which deals in how the DNA is being regulated. And there are mechanisms of opening and closing DNA. And please remember, if the DNA is open, the DNA is readable. If the DNA is closed, the DNA is not accessible. That changed the phenotype. That changed a lot of different things. So one of the interesting part is that the control over monocytes, dendritic cells, and microphages differentiation is influenced by the system called MCS. And that system is under tight control of the several regulatory elements. And one of them is the transcription factor P1, which has this small on and off switch, which we call the SP1. And that SP1 is the epigenetic switch. That means, depending on the methylation and demethylation, and those things correspond to switching on and off, this switch can be activated or not. And if you follow this diagram, this is one of the situations that you may have a positive feedback loop. Because if the MCSF is produced, it's going to stimulate P1 to be produced more. And if the SP1 switch, which is the inhibitor, is switched off, then guess what happens? Then we've got even more MCSF. And the whole process can go in the circles over and over and over and continue to produce monocytes and microphages, not dendritic cells, even though the primary corp will disappear. So this is the power of the epigenetic. Not only you may have a positive feedback loop, but this positive feedback loop may persist for a long time, days, weeks, months, after resolution of the initial insult. So now the question is, how do we design the experiment that will be most controllable and allow us for showing this persistent immunoaberrancy? I'm sure you can appreciate that any longitudinal long-term experiments, when you have to study pre-existing baseline condition of the patients, are complex. They're actually prohibitively complex. And I have to come up with new ideas. And I'm going to show you how I decided to prove my hypothesis on the next slide. So what I did, I realized I have access to actually a very good insult. By working the HVICU, I frequently witness my patients coming from the heart surgery. And I just realized, heart surgery is great. It's an iatrogenic insult. And what it allows me is to look at the patient baseline immune system performance before the surgery. Then I have a very controlled, very measurable, very quantifiable insult. And then I can follow up my patients for almost unlimited period of time. Because the feature of my health care system is that we're very vertical and horizontal integrated. So a lot of my patients stays together. So what I call over here as a long-term follow-up, it's three months. And our experimental design looked the way that we draw the blood of the patients before the surgery. Then we did look at the inflammatory response within the 24 hours right after the surgery. Then we look at seven days, which represent in our world acute resolution of the inflammation. And then we look at the recovery at three months. And our working hypothesis was that in some patients, not all of them, you're going to have a macrophagous predominance because of this epigenic abnormalities in the differentiation of the myeloid lineage. So let's talk about results. Are you guys ready? I'm very proud of my team. I'm very proud what I achieved in several years. And I'm very proud of SCCM, because all this research is practically enabled by the SCCM Vision Grant. So let's talk about the results. So let's talk about the results, the best part of our research. So let's go over the experimental setups. I'm drawing the blood at the baseline before the surgery. Then 24 hours after, seven days, which in our hospital coexist with time when the patient is usually discharged to the hospital. And then we are looking at three months. At the three months, most of my patients, 90% plus, are walking, talking. They have a normal life. At each time, we're drawing the blood. And we're using negative separation technique to obtain the monocyte samples. And what you see is the flow cytometry testing on those samples. Among many markers, we use the MRP8. And the MRP8 is the marker which is related to the macrophages predominance. So what you see over here is a situation when you see increased percentage of the macrophages. And I want you to look at the data very carefully, because what you see over here is changing in the mean and median. But the standard deviation is significant. But because we compare the data always to the baseline, this allows us to reduce inter-individual variability significantly and actually extract this data in such a form that actually shows the difference. That was not achieved in any studies before that we know. Second thing is, even though we have more macrophage-like cells as signified by the percentage of the MRP8 cells, what we also see is that those positive cells are not very professional. And this professionalism is measured by how many receptors is on the surface of that cell. So MRP is the scavenger receptors, typical macrophage receptor. And even though you have a more cells which look like a macrophages, they don't really have that much of the receptors. So those monomacrophages can be designated as immature, unfunctional, imperfect macrophages. If you remember, we're talking about a situation that we have a plant of the macrophages and we don't have enough dendritic cells. And what we did in the next experiments, we took this peripheral monocytes and we tried to make them in the Petri dish into dendritic cells using the IFR and GM-CSF. And what we discovered was that there is a significant depletion of that ability of the monocyte to become dendritic cells. But the interesting part is that it seems like it's almost even out at the three months. But when we look at the data carefully on the case-by-case basis, we find there are two population. And one population are the patients who can recover the dendritic cells potential very well. But there's also a population of the patients which cannot do that. And those patients are in the figure C. Now, this inability of the monocyte to become dendritic cells in case of the cells is not just some kind of inherited feature because our control, our healthy control individuals gave us monocytes that we can also try to do the same thing. And what we did, we tried to make monocytes out of the healthy control over the time with the three months break between two blood draws. And what you see is the significant inter-individual variability, but very little variability over the time, which tells us that ability of the monocyte to become dendritic cells is a feature. But that feature can be altered by the surgery as you see on the graph B and C. So summarizing, now we have a situation that we actually were able to demonstrate that three months after the surgery, you may have a plant of the macrophages and you may not have enough dendritic cells at least in some patients. So the question is, how did that happen? And the way it happens is actually relatively simple. When you talk about the word, you know, control the monocytes differentiation, we already mentioned the molecule called MCSF. And MCSF is something which prevents monocytes to become dendritic cells. But at the same time, it's a great promoter of the monocyte to begin to become dendritic cells. So logically, it made perfect sense, right? Well, it made perfect sense to us after we checked several other cytokines because of course, initially, we had to actually go through the couple hypothesis and see what will happen. So what we did, we actually were able to show on the graphs that if you ask monocytes to produce dendritic, to produce MCSF in response to the LPS or to response to the PMI and myosins, they always seem to produce more of that cytokines three months after the surgery as compared before. Interesting, at the same time, the density of the receptors for the MCSF was increased. So not only the cells produce more MCSF, they're also more sensitive to the effect of the MCSF. So now you have a mechanism which allows you to demonstrate that the cells don't have a choice because there's too much MCSF, they have to become macrophages and their ability to become dendritic cells are really at the disadvantage. So how did that happen? So let's go back to the, you know, pretty much essence of the talk. Now we go back to this graph, which demonstrates, oh, okay, there is this positive feedback loop and that positive feedback loop is technically turned on by this methylation and demethylation of the SP1 agent. So what we did, we actually look at this whole cycle and we said, okay, let's just look at the P1. This is the promoter in front of the MCSF and that would make the MC being produced. And as you see, we have a significant differentiation of the MCSF expression in our monocytes, but those monocytes became almost uniform positive for the MCSF. Okay, at three months. What was even more interesting, there was more P1 present in those cells expressed by the P1. So again, you have a more P1, more positive cells for the P1 and the most fantastic part is why is that? So what we look, we look at the methylation of that promoters and that methylation was actually decreased. And if the methylation is decreased, now the switch is in the permanent disengaged position. And now we're making a lot of MCSF. So this is one example of the epigenetic dysregulation, which can persist into the months after the critical care insult, in that case, cardiac surgery, and it may affect the fate of the monocytes with all the health consequences to follow. Okay, so we talk about cardiac surgery and we demonstrate I think quite convincingly that epigenetic abnormalities in the, some gene promoters can actually semi-permanently change how the monocytes or the dendritic cells are determining their fate over a long period of time after critical care insult. But these are the only conditions. So it's actually not. We conduct similar experiments in a couple other settings. One of them is actually we look at the humanized mice, which underwent sepsis, and we make the sepsis the way that they survive 20 days. And what is interesting is that we observe the same phenomena. That means that there's some dendritic cells to monocytes, monocytogenic cells blockage, and that blockage can be resolved by the MCSF neutralization. And if you read our paper, you will see that actually P1 was a culprit responsible for that. So in the sepsis, you may have a similar condition as you've seen in the cardiac patients. Finally, cardiac patient or sepsis are not the only situation. We actually induce the bacteremia in the patient in the fruit flies. So now we're talking about the fruit flies, not humans, not rats, not mice. And what we found is the several genes that were different regulated epigenetic style 28 days after we infect those fruit flies with the bacteremia. And the interesting part is that that just tells you how conservative are the epigenetic mechanism. And that's actually not that surprise. Epigenetic mechanism are relatively conservative. The one that I just described to you is the P1. And the P1 is one of the oldest actual regulators of the monocytes, microphages, and bone marrow in general. So we actually single out the mechanism which was well known, well described. And on top of that, we see that mechanism in the several different condition. Again, that just shows you that epigenetic abnormality in the way how our body regulates immune system can be actually responsible for a lot of recovery phenomena in the aftermath of the clinical calcepsis. All right, let's take the deep breath. We're almost done. I'm just going to summarize what we talk about it. And well, I think I convinced you a little bit at least enough that you can understand what I'm talking about. I convinced you a little bit at least enough that you can look into the literature that there are new immunostasis which emerge after the critical care illness. Even more interesting, this immunostasis, this different way how the immune system perform can be self-sustained. So you may have a trauma, TBA, several other conditions, but even though the culprit disappear, you still have a immune system which is malfunctioning. I also gave you a little bit taste and we'll look into the future, but epigenetic controls are kind of critical for that self-sustained processes. Now, when I say critical, please understand, this is what I'm passionate about. There are other mechanisms that are also important, but I like my epigenetics. So think about that epigenetic, think about our work, think about how you can contribute to the field. And I hope I will see a lot of papers from you in the future. Finally, I would like to thank all my collaborators there. I'm just a facet to the, a lot of people who make it happen, a lot of my postdoc, a lot of my undergraduate student, a lot of my fellows and residents. I also very appreciate the Department of Anesthesiology and Critical Care and its leadership in providing me the space, time, and the funds to do this research. And finally, Society of Critical Care Medicine Vision Grant and NIH K23 Award are the foundation of that research, and they have to be appreciated more. If you guys have any questions, please contact me or reach for my literature. My emails are on all my papers. Thank you very much. I hope you enjoyed the presentation.

epigenetic mechanisms

immune system recovery

critical care insults

abnormalities in regulation

balanced immune system response

gene promoters