So, my name is Vidula Vacharyjani, and I will be presenting to you sirtuins and immunometabolism in sepsis today. I am going to start by thanking the organizing committee for inviting me for this talk. Other than the NIH funding, which funded all my research, I can't claim any other disclosures. In the next few minutes, we'll talk about sepsis in general, not a lot, then we'll talk about the immunometabolism in sepsis and delve into how sirtuins actually, one of the many things that change it, do change it. So nearly a decade, actually more than a decade ago, with Dr. Hotchkiss' group and also the European data, we know, we've known, that the patients that die of sepsis die during late sepsis, as in greater than five days into the pathology. And they've also shown us that the patients who are dying of sepsis later have these opportunistic infections and have an evidence of immunosuppression, and I have all those references given below. We all know that. Subsequent studies have actually reiterated the same point and have shown us that, yes, it actually is true that the immunosuppression or less immune responsiveness does occur during late sepsis. And I'm going to point you to the SRS1 and SRS2 endotypes of all of these studies, where SRS1 is the one with the worst outcome in sepsis, and they are, those patients are associated with immunosuppression, and of note, and we'll revisit this area later, that hypoxia-inducing factor, which is a, if one and two expressions were significantly different in SRS1 endotype, and we'll revisit that issue in a minute. So my laboratory has been engaged in studying this early versus late sepsis and what are the mechanisms, et cetera, and I'll go through some of that. So we took this early versus late sepsis issue to the mouse model, and basically, when you're a human, you don't know when the patient presented in our ED or in our ICUs, but you don't know when the sepsis actually started. In a mouse model, you do get to know that, because you make them septic. So then we studied serially the in vivo immune response profile and, you know, published this nearly eight years ago now, that, and we delineated three phases of sepsis. The early phase, we called it hyper-inflammation, where there is, the mouse becomes septic, you give a secondary stimulus, or a secondary inflammatory stimulus in the form of LPS. These mice were, there was more inflammatory response, whereas later, 18 hours or so later, there was actually, no matter how much LPS you give them, these mice were not responsive, or they were endotoxin tolerant. And this was in line, and then the third phase, we called it resolution, but we don't really know whether they resolved, or they went into chronic critical illness, because our study ended at seven days. So then, this was in line with the THP1, or the cell models of sepsis, where people have others, and we have shown that, you know, you give them first LPS, dose of LPS or endotoxin, and the subsequent doses become less and less effective. In fact, now, the hypo-inflammation, the endotoxin tolerance has become a marker, it's an accepted marker for hypo-inflammatory phase. So in other words, we showed that the endotoxin response to hyper-inflammation in vivo in sepsis transitions to a late hypo-inflammatory response, where there is endotoxin tolerance, followed by, you know, either death, or chronic critical illness, or resolution. So my lab has studied this for over a decade and a half now, that what causes the shift from hyper- to hypo-inflammation, and what sustains the hypo-inflammation, right? Because ultimately, the majority of our patients die during this phase. So I'm going to take a step back, and I'm going to talk about an immune cell. So if an immune cell is in a basal state, it takes in glucose, converts it into pyruvate by way of glycolysis, followed by acetyl coenzyme A, oxidative phosphorylation, electron transport, and voila, you have 36 molecules of ATP per molecule of glucose, right? But this is a long process, involves a lot of enzymes, and it's a relatively slow process. During hyper-inflammation, when an immune cell sees a pathogen, it gets activated, and it does not remain in the basal state, and it undergoes aerobic glycolysis, and I'll show you in a second why and how. So an immune cell sees a pathogen, it has three goals in mind, phagocytose a pathogen, kill that pathogen, and when there are cells trying to do that in an active manner, in trying to do so, the cells are going to die. So the body has to support the cell death by regenerating new cells. So in other words, you need energy to do all these processes, and you need substrate or a biomass for nucleotide synthesis in order to produce new cells, right? So this phenomenon was observed by Otto Warburg back in 1950s, and actually in proliferating cancer cells, named as Warburg effect, where the cells will actually selectively undergo aerobic glycolysis, and the reason we call it aerobic is because there is no hypoperfusion or hypoxia in general. Even in face of that, the cells will actually selectively choose glycolysis, and in the process, they will increase the reactive oxygen species, and they'll actually go and block oxidative phosphorylation, and basically increase aerobic glycolysis. Turns out that immune cells do the exact same thing. In fact, subsequent studies have shown that glycolysis is actually important for immune cell activation, maturation, survival, and immune function. So in other words, if you're an activated immune cell, taking glucose actually block oxidative phosphorylation, selectively choose glycolysis, glucose 6-phosphate convert into pyruvate, and some of that accumulated glucose 6-phosphate will then feed into pentose phosphate pathway to generate ribose 5-phosphate. Then you get increased ATPs, although less efficient than the oxidative phosphorylation. You can consume more molecules of glucose because glycolysis' shorter pathway can be ramped up easily. So you get your ATPs, you get your reactive oxygen species in the form of NADPH so that you can kill those pathogens, and you also get increased ribose 5-phosphate. You can have nucleotide synthesis. Remind you, I talked about the hypoxia-inducing factor, and I said it's one endotype. Sure enough, it's a master regulator of glycolysis, if an alpha, and this whole glycolysis process was tied to hypoxia-inducing factor. When you come to hypoinflammation, so during hyperinflammation, we know it's aerobic glycolysis driven. During hypoinflammation, there's metabolic chaos, and I'll prove that point in a second. We put this study out, we used THP1 cells and stimulated with lipopolysaccharide, endotoxin, and we studied intracellular metabolites at different time points before stimulation and up to 96 hours after LPS stimulation. And we showed during the hyperinflammatory phase, there is glycolysis, there is glycogenolysis, and pentose phosphate pathway, just like we talked about. During hypoinflammation, however, now you have accumulated omega-3, omega-6 fatty acids. You have decreased expression of membrane lipids. You have disrupted amino acid metabolism, and more importantly, you have elevation of acetyl carnitines, which Langley et al. showed us a few years ago that it's a biomarker of poor outcome in sepsis. So like I said before, there is a metabolic chaos. So what causes the metabolic chaos, right? That's what we study. So my answer is going to be sirtuins, but that is by far not the only answer, right? And so we saw that we have, our answer is going to be sirtuins, that we have seen that SIRT1, SIRT2, SIRT3, and SIRT6 are elevated during hypoinflammation, and that is actually I have shown that the SIRT1 and SIRT2 expressions decrease during the hyperinflammatory response. So what are sirtuins, right? So these are the molecules, highly conserved molecules, basically first described in yeast, and we, mammalians, have seven different homologs, SIRT1 1 through 7. These are NAD sensors. So if you remember, during glycolysis, there is NADH, NADPH, and NAD accumulates during the Warburg effect, and so these are NAD sensors, so as soon as the NAD accumulates, the sirtuins are expressed, or they come to play. SIRT1, SIRT6, and SIRT7 are nuclear, SIRT3, SIRT4, SIRT5 are mitochondrial, and SIRT2 and SIRT2 lives in the cytoplasm, although it can translocate into the nucleus. These are legitimate anti-inflammatory and anti-aging agents, and they're implicated for the deficiency of those, implicated in all of those pro-inflammatory effects that are not all, but most of it, in aging and obesity. So how are they connected to us, right? So we showed, this was, again, what happens to these sirtuins in hyper-inflammation. I showed this, others and I, we have shown this, that SIRT1 1, which is one of the most studied of the sirtuins, it decreases during, its levels decrease during the hyper-inflammatory response, and so we have shown this in the human cells from patients and, as well as the laboratory models. And in fact, we and others have shown that if you treat mice with resveratrol, which is a SIRT1 activator, or other SIRT1 activators, it actually improves sepsis survival through multiple anti-inflammatory pathways. In the interest of time, I will not delve into details, but these were all studied mostly in the pretreatment arena, which is, we know, that clinically has limited relevance. Similarly, SIRT2N2, which is an emerging role, and that's been my new interest in my current NIH grants, and it, we also showed that there was decreased levels of SIRT2N2 in mice during hyper-inflammation, and SIRT2N2 over-expressing mice showed better survival with sepsis, lower inflammatory response, although the selective activator of it is not yet available. So when it comes to hypo-inflammation, what happens to SIRT2N1? So it turns out that we studied this several, several, a decade ago, and we showed that in the human THP1 cells, during the endotoxin-tolerant hypo-inflammatory phase, there is increased SIRT2N1 expression, and there is, when we went and inhibited it by EX527, a selective SIRT1 inhibitor, we reversed the endotoxin tolerance. Subsequently, we showed that the SIRT2N1 actually is responsible for inducing SIRT2N6, which is a regulator of glycolysis and fatty acid oxidation, as mentioned, and also SIRT2N1 is, induces SIRT2N3, which is important for mitochondrial biogenesis. So we took this about eight, nine years ago to the mouse model, and we showed that during the hypo-inflammatory phase, the in vivo hypo-inflammatory phase, we showed that there was increased SIRT2N1 expression in different tissues, intestine, liver included, innate and adaptive immune cells, and cardiomyocytes. And in a couple of publications, we ended up showing that SIRT2N1 inhibition during hypo-inflammation, only during hypo-inflammatory response, it improves mouse survival. It reverses endotoxin tolerance and improves cardiac function. So as you can imagine, SIRT2N1, SIRT2Ns, you can imagine them as homeostatic regulators in the sense that they are decreased during hyper-inflammation, but increased during hypo-inflammation. So then coming to SIRT2N2 and hypo-inflammation, that's been my latest or last five or six years I've been studying these. My first thing I'm going to talk about is the obesity and sepsis. That's what my earlier RON was studying. It's a common comorbidity. We know our patients are obese. So there is prolonged hypo-inflammation during obesity with sepsis, and we did not observe increased SIRT1, but we saw an increased SIRT2N2 expression, and in fact, SIRT2N2 inhibition during hypo-inflammation reversed hypo-inflammatory response and improved mortality. And SIRT2N2 is a known immune repressor subsequently. Subsequently, we also showed that high-fat exposed immune cells, SIRT2N2 actually dysregulates autophagy, which is critical for cell survival. And the latest one being ethanol use disorder, which is another comorbidity. We showed a couple of years ago that SIRT2N2 increases during early, even hyper-inflammatory response in mouse model of ethanol with sepsis, and SIRT2N deficient mice with ethanol with sepsis have actually, they are protected from sepsis mortality and improved bacterial clearance. We subsequently are showing that SIRT2N2 deficiency actually improves glycolysis, phagocytosis, pathogen clearance, and survival, but I'll not go into details of those because we have two other presentations describing those. Dr. Roichaudhuri will present a star research symposium this afternoon, and there is a research snapshot tomorrow, as mentioned. So with that, I'd like to acknowledge my lab here in Cleveland Clinic, as well as in Wake Forest, my collaborators from Cleveland Clinic and Wake Forest, and of course, the NIH grant, without which none of this would have been possible, and thank all of you, last but not the least, and open it for questions in the end, towards the end. Thank you.

sirtuins

immunometabolism

sepsis

hyper-inflammation

hypo-inflammation