Hello, everyone. I'd like to thank the organizers of this session for inviting me to give this talk today. My name's John Riley, and I'm an assistant professor of medicine at the University of Pennsylvania, and I've been asked to talk about a new set of eyes to study sepsis mechanisms in human organs. And this is part of the larger session, Immunology of Sepsis in the Post-COVID-19 Era, What Have We Learned? And what I'd like to do is briefly talk about some of the challenges in developing therapeutics for sepsis prior to the COVID-19 era, and how COVID-19 may have accelerated and changed our progress through a new set of eyes to study sepsis mechanisms in human organs. I have no disclosures relevant to this talk. I do receive research funding from the National Institutes of Health and the Department of Defense to study the acute respiratory distress syndrome. Now, we all know that sepsis is the dysregulated host response to infection that results in life-threatening organ failure. This dysregulated host response is broad and heterogeneous, and can result in various organ dysfunctions from ARDS to acute kidney injury, thrombocytopenia, cardiomyopathy, neurocognitive dysfunction, among others. And current therapies for sepsis appear to have improved mortality in recent decades, but remain largely supportive or focused on pathogen eradication, including early antibiotics and source control, resuscitation, and organ support with mechanical ventilation and dialysis. Unfortunately, improvements in sepsis care are not attributable to any specific therapeutic, despite many drugs that demonstrated promise in animal models, only to fail in subsequent clinical trials. And we have a long list of drugs that have been tried in sepsis that had a lot of promise that really showed no consistent benefit. So going back as far as the days of high-dose corticosteroids, which showed no consistent benefit. And then moderate-dose corticosteroids may benefit patients and certainly get them off vasopressors quicker, but there's a lot of variability across trials. IL-1 receptor antagonists and anti-TNF therapies specifically targeting specific pathways in the host immune response have not shown consistent benefits. And then ibuprofen and activated protein C targeting coagulation has also shown no consistent benefits in early sepsis trials, leaving us really to have this long list of failed therapies in sepsis. But this all changed with COVID-19. So in COVID-19, the drugs that targeted the immunologic host response, not the pathogen, have finally found success. What we've seen is that yes, there are some drugs that inhibit viral replication, and they may be able to, if given early, prevent the development of severe COVID-19. But there's been real success over the last two years in targeting the host immune response in patients that already have severe COVID-19. And severe COVID-19, to all of us, is a form of sepsis caused by infection with SARS-CoV-2 virus. And what we've seen is success with dexamethasone in hospitalized patients with COVID-19 in the recovery trial. Baricitinib plus remdesivir for hospitalized adults with COVID-19 in the ACT-II study group trial. And interleukin-6 receptor antagonists, such as tocilizumab, in critically ill patients with COVID-19. Now that we've identified several drugs that benefit patients with one form of sepsis, can we take this success and bring it back to a broader population of patients with sepsis outside of those infected with SARS-CoV-2? But there are a lot of challenges to try to apply what we've discovered in COVID-19 to the larger population of sepsis. And those problems start with sepsis heterogeneity. We've talked a lot about this, but there's heterogeneity in the host. There's heterogeneity in the pathogens themselves, where SARS-CoV-2 infection is not the same thing as E. coli infection. The infectious source, whether it be the lung or the gut or the GU system, likely introduces heterogeneity, as well as what other treatments the patient's receiving at the same time, whether it be fluids or if the patient's receiving chemotherapy for cancer, for example, etc. We also know that while our animal models have really helped us understand mechanisms that underlie sepsis, they're limited. The animal models are poorly predictive of what drugs would be beneficial to patients. There's many therapeutics that can treat sepsis in a rodent model that does not end up being beneficial to patients in large clinical trials. We also have limited cell culture models where a single cell culture of endothelium or epithelium do not fully model an organism. We also have a syndrome that's very dynamic. Sepsis has a dynamic immunopathogenesis, where over time things change and a treatment that might be beneficial at one time point may not be beneficial at another time point. We also know that the causes of sepsis outcomes, of poor sepsis outcomes like death, are not always determined specifically by sepsis and often can be a marker of the end stage of an underlying disease. So one of the ways we can address some of these problems is by using a new set of eyes in human organs. And there's a whole bunch of new ways that we're studying sepsis in novel and innovative ways, but I'd like to highlight three ways, systems that we can use to study human organs in sepsis. The first is organs on a chip, which are a microfluidic device to allow us to study injury in a sepsis model. Ex vivo lung perfusion of lungs to study acute lung injury. And then using the patients themselves as a model for sepsis through using patient biospecimens and cohort studies to understand patient heterogeneity and potential heterogeneity of treatment effect. So I'll start by talking about organs on a chip and I'm going to specifically talk about lungs on a chip because I think it's really fascinating. So what these lungs on a chip are is they're a physiologic system built on a microfluidic chip to create a microenvironment that simulates the organ in terms of tissue interfaces and mechanical simulation. And if you can think about the things that go on in the lung, you have the alveoli, which have epithelial cells. You have the vasculature, which have pulmonary endothelial cells. And then you have breathing. You have the mechanical motion of breathing, which stretches and de-stretches the cells of the lung. And what that can be created in a small microfluidic device, such as the one drawn in this cartoon here. What you can see here is that they use a small thin membrane. And on one side, they culture alveolar epithelium. And the other side, they culture pulmonary vascular endothelium. And the top chamber, they can run air through and they can have different things in that air if needed. And then on the bottom chamber that abuts the endothelium, they run blood that can also include that can also include inflammatory cells. And then what's unique about the lung is because we breathe and the lung stretches, they create these side chambers that have a vacuum that can intermittently contract and stretch the cells and then release and let the cells come back to normal. And this model can then be used to test all kinds of exposures, whether they be therapeutics or air pollution or certain inflammatory cell activation. And in one case, in some of the earlier experiments, they were able to demonstrate that if they put E. coli and cytokine mix into the alveolar space in this model, and then they ran blood with neutrophils through the blood chamber, they could actually see the neutrophils transmigrate across the alveolar capillary barrier and engulf the E. coli. So this is really a great model of the lung and how the lung is injured in setting of sepsis. And the lung is not the only organ on a chip that's been developed. There are organs on a chip developed for the heart, the kidneys, the eye, the gastrointestinal system, and the brain. And these can all be modeled in different ways that could have potential relevance to sepsis. Now the next model that I'd like to talk about that could model our organ dysfunctions is ex vivo lung perfusion. So ex vivo lung perfusion was developed for lung transplantation, where they recognized the problem that many lungs that are offered for transplantation are too marginal to be transplanted. So what the idea of ex vivo lung perfusion was is to try to rehabilitate these lungs so that they can subsequently be transplanted. They're rehabilitated by being connected to a ventilator and ventilated, as well as being perfused by either a perfusate or blood itself. And then they try to improve the lung enough that over time it can be transplanted. Now interestingly, many of these lungs still can't be transplanted despite being on the EVLP system, but they could potentially be used as an experimental model for therapeutics that are targeting acute lung injury. So one of the places where we've leveraged the EVLP model in my own research is to try to further understand the relationship between the ABO blood types and the acute respiratory distress syndrome in critical illness. So we previously reported that blood type A was associated with approximately a 14% increased risk of ARDS in the setting of trauma and sepsis relative to blood type O. And what the ABO glycans are is they're carbohydrate modifications, and they're at the end of multiple different carbohydrates on multiple different cell types, not just red blood cells. And they're actually all over the endothelium. They're also on the von Willebrand factor molecule, and they're on platelets. And we had identified this association in large cohort studies of patients, but we wanted to see if we could create a model to test potential therapeutics targeting the ABO biology by using the EVLP system. So we started by asking the question, does blood type impact the ability of lungs to improve on the EVLP system? So we leveraged the novel lung trial, and this is thanks to a collaboration with Ed Cantu at the University of Pennsylvania. And this was a trial that was a prospective multicenter cohort study of outcomes after lung transplant from marginal lungs treated with EVLP. So they took lungs that did not meet criteria for EVLP, did not meet criteria for transplantation, treated them with EVLP for four hours, and then they had to meet specific objective criteria to be eventually transplanted. So they had to have two consecutive PaO2s greater than 350 millimeters of mercury on 100% FiO2. They had to have improving or stable PVR compliance and airway pressures, and then they could be transplanted into a donor. And out of the 102 EVLP donor lungs included in this cohort study, 57 were transplanted and 45 were not between 2010 and 2014. And interestingly, when you look at ABO blood type, there was a strong association between ABO blood type and the likelihood of the lungs to recover on EVLP, whereby only 38% of blood type A lungs recovered, while 67% of blood type O lungs recovered. And this was also not just whether they were transplanted or not, but the last PaO2 on the EVLP system was statistically significantly higher in the blood type O lungs relative to the blood type A lungs. So this suggests that we can potentially use the EVLP model as a model for lung injury with relevance to the ABO risk factor for ARDS. Now the relationship of ABO blood type and sepsis and acute respiratory distress syndrome has been brought back to our attention in the COVID-19 pandemic when early reports reported an association between ABO blood type and COVID-19. And then when we have eventually had genome-wide association studies, we identified that the ABO locus was a major locus associated with severe COVID-19 respiratory failure. And interestingly, when they hone down on that locus, it's actually the genotypes that determine blood type that were strongly associated with COVID-19 respiratory failure. So that brings us back to the potential link of ABO blood type and sepsis outcomes. So the EVLP model, because it has relevance to ABO blood types, might be actually a model to test novel therapeutics. And one potential novel therapeutic is a therapeutic aimed at von Willebrand factor. So about 30% of the variability in von Willebrand factor levels in human populations is determined by ABO blood type, where blood type A has the highest levels of von Willebrand factor and blood type O has the lowest levels. It's believed that the von Willebrand factor molecule itself has ABO glycans located at the binding site of atoms TS13 that breaks down a von Willebrand factor multimers to make them less coagulopathic. Now in patients that are blood type A, the glycan actually interferes with the efficiency of the atoms TS13 molecule, resulting in higher levels of von Willebrand factor. Now it's possible that a specific therapeutic aimed at breaking down von Willebrand factor may be more effective in blood type A, and we could potentially test this in an EVLP model. Now the last new set of eyes to study organ dysfunction and sepsis that I'd like to talk about is patient biospecimens and letting the patient be their own model. And there's really been a breakthrough in this field of studying sepsis, genomics, transcriptomics, proteomics, biomarkers, all related to trying to identify what population would benefit from what therapy. So really honing in on precision medicine. And again I'd like to give an example of a place where this may have relevance. This is work led by Nula Meyer at Penn, who studied the mortality benefit of recombinant human interleukin-1 receptor antagonist for sepsis based on baseline markers of activation of the IL-1 pathway. So what she did is she obtained plasma from a phase 3 human study conducted in the 90s where they were testing recombinant human IL-1RA, also known as anakinra, as a potential therapeutic for sepsis. So in that original study there was no benefit to anakinra. However, Nula hypothesized that if you stratify by baseline level of IL-1RA or IL-1B, you might be able to identify a population that would benefit. So patients that have the higher level activation of the IL-1 axis may be more likely to benefit from an IL-1 inhibitor. And she found exactly that. So if you look in this curve, this Kaplan-Meier curve, what you can see is in red are the patients that had plasma that had the highest levels of IL-1 receptor antagonist. And if they also received the recombinant human IL-1 receptor antagonist, they had a lower mortality than if they received placebo. Now in the patients that had low plasma IL-1 receptor antagonist, they had a lower overall mortality because they had less marker of inflammation and were less sick. But they did not see a benefit to therapy with recombinant IL-1RA. And if you look specifically at the numbers, based on a cutoff point that was derived from the data itself, if they had a low level and got placebo, their mortality was 18% versus 26% if they received recombinant interleukin 1 receptor antagonist. But if they had high levels of activation of the IL-1 axis, the placebo mortality was 45% versus 35% in the IL-1RA treatment group. And this was statistically significant both within the stratified analysis and the interaction between the two groups. So in conclusion, developing therapeutics aimed at the host response in sepsis remains a challenge. Novel organ models may provide new insights into potential treatments. And novel organ models and patient specimens may help identify those most likely to respond to those potential treatments.

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