My name is Tony Hernandez, and I thank you for inviting us to share our work this year involving trained immunity and how it may impact the outcomes of perioperative and critical care medicine. I have no financial disclosures, and most of the work I'm about to show you is supported by this grant from the National Institute of General Medical Sciences. Our objectives are to summarize the rising threat of microbial resistance, to define the role of toll-like receptor 4 agonist as a mechanism for inducing trained immunity, and lastly, to explain the role of trained immunity in models of perioperative medicine. We're going to go ahead and start with the first objective in talking about the rising threat of microbial resistance. In this publication in JAMA, they describe how in the United States there's a rising problem with antibiotic resistance. In this publication from 2016, they nicely describe here on the x-axis, you see the number of years from the time that the drug class or specific drug was developed, and here you see the timing of the emergence of the first reported resistant strain. It's a little bit sobering to identify the majority of them are within five years. Now vancomycin did take longer than 30 years, but still the majority of the classes are within five years. This publication by Tracy Hampton describes how by 2050, we anticipate having over 300,000 deaths in the United States alone from infections that we simply won't have antibiotics to treat with. Traditionally, we have depended on pharmaceutical companies to come up with new products that will help us neutralize this host pathogen. However, pharmaceutical companies are now identifying that this is no longer viable, and startup companies such as the one led by this physician here in the picture have gone belly up because it's simply not viable to do so. So the strategy that we have depended on in the past may not be one that we can depend on moving forward. As a result, Washington has taken note of this and offered a multitude of incentives to help identify novel programs to combat this antibiotic resistance. So now we'll talk about the role of tolic receptor for agonist as a mechanism for trained immunity, since trained immunity may be a mechanism to deal with this problem. During this presentation, I will go over innate leukocytes, and I want to just describe that there are two types. There's phagocytic cells and non-phagocytic cells, and the ones I will be describing are neutrophils, monocytes, and macrophages. The dogma has been that innate immune cells cannot build memory to pathogens, rather that they respond rapidly, neutralize the pathogen, present the antigen to the adaptive immune cells, which then develop memory. And this takes anywhere from 72 hours to maybe a week, but they do have memory. In the last decade or two, we've learned that that's not true. We've learned that innate immune cells, in fact, can develop memory, and that these phenotypes, after being primed with a pathogen product, essentially have induction of innate immune memory, which we now just term trained immunity. When somebody asks me for one-liner, like in an elevator, I'll just say, well, trained immunity is essentially inducing innate immune memory against pathogens. One of the mechanisms of doing this is activating toll-like receptor proteins. Now I chose to work with TLR4 because it has two pathways. It works through the MITEI88 pathway, which influences inflammatory cytokines, and also the TRIF pathway, which influences interferon production. Here you have a picture of what is LPS, or native E. coli lipid A. You notice that there are two phosphoryl groups and six acyl chains. Each of these chains can be anywhere from 12 to 16 carbons in length. By cleaving one of these phosphoryl groups, you end up with monophosphoryl lipid A, and by doing so, you reduce the toxicity to humans by just over 100 fold. In fact, many of us have probably received MPLA. I know I have. MPLA is derived from Salmonella Minnesota strain 595 through a series of hydrolysis reactions. These are some of the vaccines that contain MPLA, and typically it is bound to an aluminum salt called alum. And so I did not list the numerous cancer vaccines that are currently being studied. However, MPLA in itself is not manufactured by GSK for use other than a vaccine adjuvant, and therefore we're still searching for a product that might help us easily induce a trained immunity that would be safe for humans. So how can trained immunity be used to prevent infection? Well, in previous studies using animal models, and here is a simplification, for example, a mouse might have received MPLA, whether it be intravenous or through the peritoneal cavity, and its control would receive saline. Then, for example, an infection, and this is something that was done in our laboratory where we induced abdominal sepsis through cecal ligation and puncture, then we followed the animals for survival for seven days, and the animals who received MPLA had approximately 75% survival versus those animals who were treated with saline did not survive. And this is an opportunity that we see with a product like MPLA that it would be manufactured that is safe for humans and how it could be employed to prevent infection. Now here you see, again, this is the picture of E. coli lipid A, monophosphoryl lipid A, and down here you see these ultra-pure synthetic, essentially clones of MPLA. They're manufactured in the United States, and I'll show you a close picture of it, mainly to show you that this product, which is the lead for pharmaceutical development, only has five chains, and actually it's the lack of that fifth chain that makes it a higher candidate for pharmaceutical development. But we sought to test whether these ultra-pure synthetic compounds perform like what appears to be their clone, MPLA. Again, just as a reminder, MPLA signals through TLA receptor 4 protein down the mighty ADA pathway in TRIF, and so we wanted to test how these fads performed compared to MPLA and a negative control. And what we found here is, what we're looking at, this is a western blot, and we're looking at bands here that represent phosphorylation of the IKK proteins, which should be down the mighty ADA pathway, and then IR3 phosphorylation that represents the TRIF pathway. To the right here, because we can't really tell just by looking at the bands how dense they are, we employed densitometry, and we're able to plot the density of those bands in a bar graph. Now, here you see, and by the way, we used a bone marrow-derived macrophages to test the phosphorylation. And so we see the wild type and the TOR-like receptor for knockout mice. So for these mice, we see no phosphorylation, and that's what we would expect. Whereas the fad and 3D 6-acyl fad show significant phosphorylation for both IKK and IR3, 3D fad does not show that degree of phosphorylation. Nevertheless, we wanted to see how well it performed. So we're going to move on now to explaining the role of trained immunity in models of perioperative medicine. Again, I want to remind you that these animals did not receive any antibiotic for these infections. So this particular experiment involved intraperitoneal administration of fad or MPLA or vehicle, the control group, at 48 and 24 hours before the intraperitoneal injection of Pseudomonas aeruginosa 1x10 to the 8th. At six hours, we euthanized the animals, and we wanted to collect the peritoneal lavage fluid to measure bacterial burden in cytokines as well as blood for cytokines. And what we learned is that those animals that were treated with a control, with vehicle, ended up becoming hypothermic, but all the other treated animals maintained their thermoregulatory properties. When we looked at the bacterial burden in the peritoneal fluid at six hours, all the animals that had been treated with either MPLA or any of the fads demonstrated about a 50% reduction in Pseudomonas aeruginosa. Then we looked at the cell types that were responsible for or present in the peritoneal cavity, and we found that neutrophils and monocytes were particularly increased as compared to the vehicle animals. Next, we wanted to identify what the production of cytokines were. So here you see that the vehicles produce a higher amount of cytokines as compared to the treated animals. And the reason we were interested in looking at this is because cytokines oftentimes correlate with mortality. Now thus far, we used a product that resembles a gram-negative bacterial cell wall, and therefore we would expect that there would be memory against gram-negative cell pathogens. The question now is how well will it perform against gram-positive organisms? So to answer this question, we went ahead and treated the animals in a very similar fashion except this time we gave intravenous fad or MPLA at two and one day before an intravenous dose of one times 10 to the eighth of Staphylococcus aureus. I hope you take note that we're using organisms that often plague us in the ICU. Now this was a survival study, and what we identified is that there was at least a 50% to 60% survival benefit in any of the treated animals. Now normally in the ICU, we don't see a bolus of Staphylococcus aureus. Usually infections from central lines are more progressive. So we still found that this was quite impressive. We then wanted to learn if this had to do with bacterial clearance. So to do that, we went ahead and did the same treatment as described before, same dose of infection with Staphylococcus aureus, only this time we euthanized the animals at 72 hours. And the reason we did that is beyond 72 hours, the animals began to die from septic shock. So we wanted to capture as many of them as we could. And what you identify here, or you can see, is that there's an increased bacterial burden in the lungs, spleen, and kidneys of those vehicle animals as compared to the MPLA or fad treated animals. The next question we had is, is there anything different about the trained innate leukocytes that improve survival and reduce bacterial burden? So we sought to explore if there was enhancement in phagocytosis. And so here we identified that there is, in fact, enhanced phagocytosis in the neutrophils, monocytes, and macrophages of the treated animals. We then wanted to know if a respiratory burst was also enhanced, and we found that that was also the case in neutrophils and monocytes. So not only does fad treatment and MPLA bring more leukocytes to the site of infection, but it enhances its antimicrobial properties of these specific leukocytes. Now a question that's been asked for us is, how does this work in other models of injury, not just infection? So here we sought to test if fads actually protect against inflammatory, in this case ischemia reperfusion injury, and we used a model of acute kidney injury to study that. So we want to specifically explore if fads confer disease tolerance. Why did we choose acute kidney injury? Well, it's fairly common in cardiac and non-cardiac surgery. And in fact, for those patients who end up requiring dialysis, there is an associated increased mortality with them. So we still have an unmet need to help preserve organ function in this patient population. And so here in this graph, we basically show a relationship that as you have an increased amount of injury, you have an increased amount of organ dysfunction or organ failure. But our hypothesis is that if you train the immune system, that maybe you would have a reduced amount of injury and hopefully preservation of organ function. So we went ahead and used 3d6-acyl fad, since we know now that it does induce trained immunity. And what we did to test this hypothesis is we collected baseline blood, and then we gave intravenous vehicle or fad, in this case, we gave the dose of 200 micrograms at 48 and 24 hours before the ischemic insult. And the specific ischemic insult was a bilateral renal artery clamping for 24 hours, and then reperfusion. We then collected blood 24 hours after the ischemic insult, and then two days later, now being three days after the ischemic insult, we collected blood and harvested the kidneys. And what we identified is that there was a preservation of organ function as measured by BUN and creatinine and those animals that were treated with fad. And then when we looked at the structure of the kidneys, we noted that there's significant inflammation in casts and those animals that were treated with vehicle, but you don't see that a degree of inflammation or casts in those animals that were treated with fad. We also assessed using qPCR, we wanted to assess the amount of mRNA of N-gal in the kidney tissue. So this is kidney tissue N-gal, and that's significantly lower in the fad group, and the same with hemoxygenase, and hemoxygenase is often induced in the setting of injury as it has anti-inflammatory properties. What we did see an increase is an interleukin-1 beta, and this is often produced by macrophages. And we've learned that MPLA in particular, and we're now studying a fad, changes the characteristics of macrophages to confer changes in metabolism. So we're looking forward to exploring that. Here you have the histology score, by the way, showing the differences in the tissue. So in summary, there continues to be a resistance to antibiotic therapy, and this continues to rise globally. Trained immunity appears to be a plausible approach for antimicrobial resistance independent of antibiotic therapy. Trained immunity also appears to enhance disease tolerance, and in particular, in a model of ischemia reperfusion-induced acute kidney injury. Lastly, phosphorylated hexaacyl disaccharides appear to be strong candidates for pharmaceutical development. I thank you, and I look forward to your questions.

trained immunity

perioperative care

critical care medicine

microbial resistance

innate immune memory