All right. So I don't have any relevant disclosures other than some of the things I'm going to discuss in the next 15 minutes are funded from my research grants, including some work I'm going to talk about at the end that was funded by the Society. So my objectives are to review the role of mitochondria in sepsis-induced multiple organ dysfunction syndrome, or MODS, and highlight potential mechanisms of mitochondrial dysfunction that could be therapeutically targeted to improve patient outcomes. So the foundations of life include structure, information, and energy. And for the most part, Western medicine has been organized around structure, right? So we see that with our subspecialties, cardiology, pulmonary, renal, and so on, and more recently, the addition of DNA as the role of information becomes increasingly relevant to our care of patients. But really, the difference between life and death or animate and inanimate is energy, not structure and information. And of course, in human energy, that's ATP. And Eastern medicine is really focused more on this aspect of life, right, with the focus on the qi, or the life force, the flow of energy that sustains living beings. And humans spend a lot of time making ATP and energy that they need to sustain life. And in fact, the average human produces almost its entire weight turnover every day in the form of ATP. And most of that ATP is produced in the mitochondria through utilization of oxygen, such that human beings are kind of operating essentially at about a 60 to 100 watt bulb. And it's been known for quite some time that oxygen consumption and energy metabolism are altered in critical illness. So this study from a few decades ago now showed that with increasing severities of septic illness, there was a decrease in oxygen consumption and a decrease in metabolic rate that improved in those patients who recovered over time. And Mitch Fink demonstrated nicely in a rodent model that this is somewhat specific to sepsis compared to other forms of shock. So on the left-hand side of this graph are rodents who were made hypotensive through hemorrhage. And as you follow the graph upward, you can see progressive deterioration in blood pressure with a left shift in tissue oxygen pressure, suggesting a fall in tissue oxygen pressure with hypotension. However, if you make the rodent hypotensive with injection of LPS or endotoxin, you actually see an increase in tissue oxygenation, suggesting that there's a decrease in the ability to utilize oxygen in endotoxin that was not there in hemorrhage. Moreover, we see in human studies, we're all very familiar with the high mortality when central venous oxygen saturation is low, which usually indicates a decrease in oxygen delivery. But this is really a U-shaped relationship. And so there's a similar increase in mortality when central venous oxygenation is inappropriately high, suggesting that there's an inability to actually utilize that oxygen to produce ATP. We see a similar pattern with lactate levels, such that if we look at an elevated lactate level of about four, this has different prognostic meaning depending on the timing of when you measure that lactate. So for patients who have an elevated lactate early on in their course, there's a lower predicted mortality than if you have the same level of lactate later on in that septic illness. And that later hyperlactatemia, it's much more likely to be reflective of an inability to utilize oxygen, as opposed to solely a decrease in delivery. So that leads us to the very complex maps of human metabolism. And we're not going to get into the details of all this, of course, in 15 minutes. But we're going to focus for the next seven or eight minutes on the subset of these reactions that occur within the mitochondria. So abnormalities in mitochondrial structure and function have been known for some time, and in fact, have been documented in pretty much every organ system that's been studied in both preclinical and human studies of sepsis. And these are just some representative more recent studies. Of course, there are many, many more. And just to remind you the role of mitochondria in producing energy. So mitochondria are double membrane organelles that exist in all of our cells, with the exception of red blood cells. And embedded within that inner mitochondrial membrane are the complexes of the electron transport system, for which NADH and FADH2 donate electrons, which are passed along through the system by a series of redox reactions that ultimately reduce oxygen to water. And through that process, protons are pumped from the mitochondrial matrix into the intermembrane space, producing the chemoasmotic gradient that is ultimately used to be pumped back through complex 5 or ATP synthase to produce ATP. So if there's any disruption in this process, and there's many reasons why there could be a problem here, that leads to a bioenergetic crisis due to a reduction in energy available for the cell, which then becomes injured. If enough cells are injured, you have dysfunction of organs and ultimately organ failure. In addition, the electrons are then backed up here and then are offloaded to other species that produce reactive oxygen and nitrogen species, which further cause cellular injury and exacerbate this problem. Studying mitochondrial function has been a problem and has been a major impediment to kind of moving forward in this area. And so my lab and others have tried to get around this by utilizing a pragmatically available cell source from blood, because you need the cells to study the mitochondria. And so we look at peripheral blood mononuclear cells, which are circulating monocytes and lymphocytes. And we measure oxygen consumption through high-resolution respiratory. And in a pilot study about 10 years ago, we were able to demonstrate that maximal oxygen consumption and spare respiratory capacity, which is a measure of the ability of a cell to respond to a metabolic stress, were both impaired in septic children compared to healthy controls. We went on to replicate this finding in a much larger study with a much more heterogeneous set of patients with different illness levels of severity, again showing that same decrease in oxygen consumption that improved as most of these kids got better over time. And what we had hypothesized initially was that we would see a association between early severity of depression of mitochondrial oxygen consumption with illness severity. And in fact, we did not see any of that. And perhaps that, in hindsight, could have been predicted by some of what you just heard in the prior talk, that some of the shift and downregulation of mitochondrial oxygen consumption probably reflects normal immune activation. But on day one, there was no association between oxygen consumption in these cells and PILOD score, which is the Pediatric Composite Measure of Organ Dysfunction Severity. However, we did see a relationship emerge in that, over time, the children who continued to have higher level of PILOD score had lower level of mitochondrial oxygen consumption. And so in a post-hoc analysis, we went back and actually regrouped the patients, not by how they presented initially, but rather how they progressed over time. And we found that those patients who had a failure to recover mitochondrial respiration within PBMCs had a much slower rate of recovery from their organ dysfunction. And this was not statistically significant. We were not powered for this initial analysis, but certainly was suggestive that there was something going on here. And so in thinking about why this might be physiologically relevant, we have to remember that mitochondria have bacterial ancestry. And so they retain many of those properties, including the ability to replicate through a process called biogenesis. They can fuse to consolidate good functioning elements of the mitochondria. They can fizz to isolate dysfunctional elements. And then they can be selectively broken down through a process of mitophagy, so that in total, you're able to maintain a pool of functional mitochondria that are built up by biogenesis and broken down by mitophagy. And when you have a septic illness that causes some dysfunction within a partial pool of your mitochondria, if you can't replace them with biogenesis and break down the dysfunctional mitochondria with appropriate mitophagy, then you're left with a pool of dysfunctional mitochondria. And so there is some evidence that mitochondrial biogenesis is important to rescue the mitochondrial phenotype in sepsis-induced MODS. And so in this study of adult patients with sepsis, we can see that those who survived had increasing genetic markers of activation of mitochondrial biogenesis. And in a more recent study looking at PBMCs in adult sepsis, there was an increase in mitochondrial mass over time in these patients. That was initially reduced on day one, but as the patients improved, there was an increase. However, it was not uniform. So those patients who were more likely to be ICU-free at one week and survive, which are in the gray dots, had greater activation of mitochondrial biogenesis and an increase in mitochondrial mass over time. We also see that there's some genetic correlate to this. So in the GLUE grant that was published in the early 2000s, and I don't have time to go into the data, but they demonstrated that there was widespread suppression of mitochondrial energy production and protein synthesis. We tried to replicate this in pediatrics through a collaboration I had with the late Hector Wong, in which we took some of his transcriptomic data in pediatric sepsis, and we looked at the nuclear-encoded mitochondrial genes. And we found that there was broad dysregulation in these mitochondrial genes compared to nonseptic controls. And about half of the genes from the electron transport chain complexes were actually downregulated. And just about all of the mitochondrial ribosomal protein gene expression was downregulated. And this is the machinery necessary to generate and build new mitochondria. We then group patients, depending on their degree of mitochondrial gene suppression, into three groups, with group A demonstrating the greatest degree of mitochondrial gene suppression. With here, each pixel represents one gene, and the color represents whether it's up or downregulated relative to nonseptic controls. So group A had sort of broad downregulation of mitochondrial gene expression, and they had, by far, the greater mortality, as well as higher level of organ dysfunction. So we went back to our study, and we said, was there any evidence that mitochondrial content or biogenesis is impaired as children recover and move through their course of septic illness? And in fact, similar to the adult data I showed you, we showed this same increase in mitochondrial content in PBMCs over time. But again, this was not evenly distributed amongst all patients. And it was only that subset of patients who had recovery of mitochondrial oxygen consumption that actually demonstrated this increase in mitochondrial content. So lastly, I'm going to end with an offshoot of how we could potentially utilize this data therapeutically. And this was a study that was funded through a Weill Research grant that I received several years ago, where we collected, in addition to the mitochondrial measurements, we collected stool from these patients to look at the intestinal microbiome. And you can see that, as expected, compared to the healthy controls on the left, the septic patients had a fallout of their mitochondrial diversity over time, with loss of the colors indicating decreasing presence of different bacteria. And that can be quantified with decreasing amounts of beta diversity summarized in the bottom graphs. But what was really interesting was the selective loss of the different types of bacteria. So here, each column represents a different patient. And again, the healthy controls are on the left, and the septic patients are in the middle, and they move over to the right over time. And you can see the loss of the green color in the septic patients. And the green color is important, because these are organisms that are representative of the Firmicutes phylum, which are organisms that tend to produce butyrate as a byproduct of starch fermentation that is not otherwise digested, and it gets into the colon. And butyrate is a short-chain fatty acid. There are others as well. And we went on to measure these short-chain fatty acids in the stool. And we did see a slight decrease in butyrate in the stool that corresponded to the time frame in which the butyrate-producing organisms were also lost from the microbiome. And butyrate's relevant, because it seems to play a role in mitochondrial biogenesis. So these are data from John Alverde's lab in Chicago that showed that, similarly, if you challenge a rodent with polymicrobial sepsis, you see a similar loss of stool butyrate that can be rescued if you give the mouse or rodent a fecal microbiota transplant that you don't see if you autoclave that transplant and therefore make it inactive. And in a separate study, if you give mice butyrate-enriched chow, you see an increase in mitochondrial biogenesis genetic markers and an increase in mitochondrial content. So this selective loss of butyrate-producing organisms from the microbiome may somehow directly impact the ability to upregulate mitochondrial biogenesis over time and perhaps prevent recovery from sepsis-induced mods. So lastly, we asked the question of whether, in vitro, we could show that butyrate could have a therapeutic potential. And so we have a very simple model where we take EBV-induced lymphoblasts, or EBV-transformed lymphoblasts, and we incubate them with LPS in vitro. And that knocks down mitochondrial respiration in these cells about 20%, which is similar to the reduction in mitochondrial oxygen consumption we see in PBMCs from our children with sepsis. So that's why we like this model. And if we add butyrate four hours after LPS exposure to cells that were controls and didn't receive LPS, we see no change and no impact of butyrate on mitochondrial oxygen consumption. However, butyrate exposure four hours after LPS exposure did result in restoration of mitochondrial oxygen consumption to levels that were at or above control levels, suggesting potential therapeutic potential in a mitochondrial-specific manner. So why is this relevant? So we know that about 2 thirds of people, both children and adults who die from sepsis, do so with persistent rather than worsening multi-organ dysfunction syndrome. And so these are data showing organ dysfunction scores in the days leading up to death. They generally don't increase, but rather just don't get better. And ultimately, most of these patients pass away from withdrawal of life-sustaining technological support when their patients are not improving. So that suggests there's an opportunity to intervene to hasten or accelerate organ failure recovery in those patients who can't do that on their own. So rather than targeting metabolic resuscitation at the onset of illness, perhaps there's an opportunity later on in the subset of patients who don't demonstrate metabolic recovery to help promote that exogenously. So in conclusion, mitochondrial alterations are certainly part of sepsis-induced multiple organ dysfunction, whether they truly cause sepsis-induced MODS or are part of a feed-forward propagation system is not yet entirely clear. However, mitochondrial recovery, perhaps through biogenesis but may also through other mechanisms, is certainly associated in both preclinical and clinical studies with recovery from MODS and may, in fact, offer a novel therapeutic target to help us improve patient outcomes, especially in the subset who fail to recover with standard therapies. So thank you very much. Look forward to any questions.

mitochondria

sepsis-induced multiple organ dysfunction syndrome

ATP production

mitochondrial structure and function

oxygen consumption