All right. Good morning, everybody, and I'd like to start out by thanking the organizers for giving me a chance to present my work and our work. So other than NIH funding, I have no disclosures for this talk. In the next few minutes, we'll talk about sepsis. This audience doesn't need any introduction. We'll talk about how immune response in sepsis transitions from hyperinflammation to persistent inflammation and immunosuppression, and then what controls it, the shift. And then we'll talk very little about the therapeutic implications and just an example of it. Sepsis is not new. We've known sepsis since 1800s. It's just 2,000 years, and we have not been able to, well, 200 years, but we have not been able to cure it, that's all. Historically, in 1900s, we defined what the sepsis was formally at the end of 1990s, and then we started studying molecular mechanisms of it, which we continued through 2000s and came up with the guidelines and increased awareness for the disease. As a consequence of all those guidelines and increased awareness of sepsis, use of bundles, et cetera, we improved the early therapies, and we improved the early or decreased the early mortality. But as a consequence, the patients now, we all can agree that they remain longer term in the ICU, and they give us a chance to see what the natural progression of this disease looks like. And the concept of chronic critical illness developed by my friend here on my left, and he's going to talk about that in the next lecture, so I will not delve deeper into that. So just in 1900s, 1980s and 90s, with the discovery of association between endotoxin and pro-inflammatory cytokines, et cetera, there was a lot of enthusiasm about the exuberant inflammation theory of sepsis, and that led to multiple clinical trials of high-dose corticosteroids, anti-pro-inflammatory cytokine therapies, et cetera, which all failed to show survival benefit. Along with that, there was a theory of immunosuppression going on, and that realm was actually led by surgeons. By their observation that the anti-inflammatory cytokine expression actually leads to sepsis in post-operative period. So what is it? Is it pro-inflammation? Is it anti-inflammation? Is it both? Is it transition? So that's where we're going to go deeper into it today. So this is now 10 years ago, seems like yesterday, that we set up this model of sickle ligation and puncture. And the reason for our experimental model is because unlike patients, now you know, you give sepsis, so you know when the initiation of sepsis was. And then we checked the immune response in these mice by way of secondary endotoxin, sort of inflammatory stimulus, and then we studied the leukocyte adhesion response in the microcirculation. Now, why leukocyte adhesion? Because that's the earliest immune response in vivo, if you like. So basically, this is what it looks like. A free-flowing leukocyte is minding its own business in the microcirculation, but it comes along an inflammatory stimulus, or it comes along your sepsis, and the cells start to slow down, called rolling, followed by adhesion, because they have to negotiate, the leukocytes have to negotiate their exit from the microcirculation into the interstitial tissue to fight infection, whether it's there or not, and then extravasation of these leukocytes. It turns out that the leukocyte adhesion part, the one that is circled by a red box, is a very faithful indicator of inflammatory response, as in more the leukocyte adhesion, more inflammation, so less the inflammation. So what we did was we made a mouse septic and gave them either normal saline or lipopolysaccharide at different time points, meaning thereby, if the mouse was, or the microcirculation was immune-responsive, then you would have more leukocyte adhesion on top of the sepsis with LPS, but if they ignored it, the mouse was endotoxin-tolerant, simple concept. So this is what we saw. What we saw was on the y-axis, there is leukocyte adhesion, on the x-axis, there is timing. So if you gave these mice, within the first 12 hours, you gave them LPS, the black bars are significantly higher than the white bars, which is sepsis alone. So if you gave them LPS, LPS can upregulate leukocyte adhesion on top of the sepsis, but if you waited for 18 hours and then gave them sepsis, basically these mice ignored the lipopolysaccharide and there was no increase in leukocyte adhesion, meaning thereby, they were endotoxin-tolerant, and that persisted for about 36 hours post-sepsis. The mice that were surviving for about 72 hours, about 40% of them, had regained a part of their responsive status, which persisted in seven days. And so we coined these terms, hyper-inflammation, hypo-inflammation, and resolution. These terms are very confusing, I know, I understand that, but the mouse hypo-inflammation is equivalent to human persistent inflammation and immunosuppression that my friend is going to talk about. So we can all agree that, at least in the mouse model, the early hyper-inflammatory response transitioned into a hypo-inflammation or persistent inflammation immunosuppression phenotype. And the last part, we labeled it as resolution, but it's really not resolution because the whole process is not resolved yet. So does this transition, sure, it happens in vivo, but does it occur in innate and adaptive immune cells? So this paper came out in 2005, where healthy volunteers were given lipopolysaccharide, and they showed that there was a self-limiting inflammatory response in these people, and there was a transient dysregulation of leukocyte bioenergetics and transcriptomics, meaning thereby there was a transient immune cell response. Now in us and others, multiple publications have shown that the monocytes from septic shock patients and other innate immune cells, they undergo endotoxin-tolerant phenotype, and in fact, endotoxin tolerance is now considered as a biomarker for immunosuppression. So then again, this transition, myeloid-derived suppressive cells have a crucial role for this transition, but that will be covered mostly in the next lecture, so I will not talk about that. And in the adaptive immune cells also, the T cells also show the immunosuppressive phenotype, and most of the work is done, or majority of the work is done by Hotchkiss group, and they showed that interleukin-7 pathway, PD-1, PD-L1 pathway, IL-17 pathways are involved in this process, where the T lymphocytes are also immunosuppressed. And also Ayala group showed a P38 MAP kinase, so multiple pathways are involved in suppressing the adaptive immune response in this hypoinflammatory phase. So sure, we saw that the acute hyperinflammation transitions into persistent inflammation immunosuppression, but what really causes this transition, what causes this pro- and anti-inflammatory response to occur, what regulates the regulators, correct? So if you take a step back from the immune cells, there is initiation by inflammatory stimulus or a pathogen response, you have activation of pattern recognition receptors, because of the pathogen-associated molecular patterns, or PAMs, or damage-associated molecular patterns that come subsequently because of tissue damage. So the pro-inflammatory cytokines and chemokines are initiated by PAMs, but then sustained by PAMs and DAMs, and the apoptosis, of course, because of all this, there is ongoing apoptosis. So all those three processes bring out pro-inflammatory milieu that is unsustainable for any organism, and so reprogramming of the immune cells happens, which looks kind of like hibernation state. So what happens during the reprogramming, right? What does this? So the very good data about transcriptomic changes is shown in about 70% or better of the leukocytes, and what happens during that programming is now you have increased pro- and anti-inflammatory cytokines, and there is decreased, so you have increase in both anti-inflammatory cytokines and pro-inflammatory cytokines, and decreased expression of antigen-presenting cells or antigen presentation, which now sets up a storm for lack of bacterial or pathogen clearance. And this phase is associated with endotoxin tolerance, which I said is a biomarker for immunosuppression. So what programs a programmer? What programs this whole transcriptomic change? And that brings me to epigenetics. So epigenetics is a sustained environmental effect on gene expression without change in DNA. So how does that happen? So if you think of your chromosome as slinky that we, the kids play with, or some adults play with it too, and so you open the slinky, so that's your open chromatin, and that open chromatin is, means it is accessible to the transcription factors, whereas if you close the chromatin, close that slinky, it's inaccessible, the genes on that part are inaccessible for a transcription, right? Simple enough. So what causes this opening and closing is your histone backbone, the four big, or eight molecules that are around which the chromosome is wound. So if you change the histones, modifications such as acetylation, deacetylation, or methylation changes this. Acetylation makes the slinky open up, and deacetylation makes it close down, and methylation or trimethylation makes it close down, and demethylation makes it open back up. And so you can imagine that what brings, that brings with the metabolism, what brings these acetyl or methyl molecules. Obviously they come from metabolism, and so now this metabolism and epigenetics are interrelated. So with the, you know, the acetyl molecule comes from acetyl coenzyme A, acetyl coenzyme A comes from your glycolysis, fatty acid oxidation, et cetera. And so in the beginning, or the initiation phase, now you have Warburg effect and you have glycolysis, but in the later immunosuppressive phase now you have a metabolic chaos. And so what are the therapeutic implications? Can all these histones be used as therapeutic targets? So we talked about this. Basically you have the hyperinflammation transitions to hypoinflammation, epigenetic changes, transcriptomic changes. We came up with a hypothesis, and hang on with me for just a second, that histone deacetylating enzyme sirtuin modulates sepsis transition from hyperinflammation to hypoinflammation. So what is sirtuin? Sirtuins are the anti-inflammatory molecules. They are nutrient sensors, so they are dependent on metabolism. And they are also deacetylators, which deacetylation leads to closed chromatin. So they sit at the junction of all three here. There are seven of these, sirtuins one through seven, and we studied only sirtuin one and two. Others have studied three, four, five in this realm. I'm just going to give you a small example of how these sirtuins can then be used as therapeutic targets. Methylation targets are also being tried in this realm. So we observed that in that same paper 10 years ago, that the sirtuin one goes up in lean mice and during the hypo phase. And if you block sirtuin one, it actually leads to improved mortality or improved survival in mice, and also reverses hypoinflammation. And sirtuin two is a known immune repressor, and we have shown now in two different models, obesity, sepsis in the late phase, and ethanol or alcohol-induced immunosuppression. We have shown both these realms, sirtuin two plays a crucial role. And if you block sirtuin two, now you show improved mortality. So that brings me to my small conclusions for now, that the innate and adaptive immune response in sepsis transitions from the hyperinflammatory to a persistent inflammation immunosuppression, and this hypoinflammatory or PII phase extends into chronic critical illness, and there is increased incidence because of the early mortality that is improved. The transition, this transition is regulated by transcriptomic epigenetic and metabolic reprogramming of the immune and non-immune cells as well. And the phase-specific therapeutic targets, with a goal to return to homeostasis, not so much as pro-inflammation or anti-inflammation, but to return to homeostasis in a phase-specific manner should be the goal. That brings me to acknowledging my lab, who actually did all this work, and I just come up here and talk, my collaborators, and of course ENIH for funding me to make all of this possible, and all of you for paying attention and open this for questions, but we'll have questions in the end. Thank you.

sepsis

immune responses

epigenetics

sirtuins

therapeutic targets