All right, so I want to thank the society for inviting us to share our work. What I'm going to talk about here is more of the perspective, what we expect to hopefully move to in the future in terms of immunomodulation. Have a couple of disclosures here. The work I'm about to show you was supported by this grant from the NIH. Also in July, we did receive a patent award on the products I'm going to discuss, however, we don't receive any financial incentive for that. Rather, the patent is for use so that we can maintain the rights to use this product and develop it further for human study in the future. So the objectives are to summarize the rising threat of microbial resistance. This was recently published again by the WHO in the last couple of weeks. Understand the role of Toll-like Receptor 4 agonist as a mechanism for inducing trained immunity and then observe to see what are the prospective applications of trained immunity and models of perioperative medicine. So I'll start off with this publication now from 2015 where they project that in the United States alone, there will be over 300,000 deaths from infectious diseases that have resistant strains where we will not have antibiotics to treat. Now this publication preceded COVID, so we believe that these projections are actually quite conservative and may actually be worse, the actual projections may be worse. Traditionally as clinicians, when we have noted emerging strains, the pharmaceutical companies have supported us by coming up with new classes of antibiotics for us to manage patients who have these resistant strains and create infections. However, due to the economic pressures in the market, especially smaller pharmaceutical companies have gone bankrupt that had focused on the development of antibiotics and the larger pharmaceutical companies have had to shift their focus outside of the antibiotic realm. So this led to Washington offering some incentives to develop novel approaches on how we can combat this antibiotic resistance problem that is truly a global threat, it's not specific to the United States. So next let's talk about the role of TLR4 agonist on inducing trained immunity and before we do that, we need to discuss what is trained immunity. Now traditionally we had been taught that innate immune cells, these are for the purposes of this talk, there's more than the ones I'm going to show you, but we're just going to be talking about neutrophils, monocytes, and macrophages. And so the thought is that these cells don't have memory, they just respond to whatever is perceived as foreign and that's it. And then that the adaptive immune system is the only system that is capable of developing memory towards a specific pathogen. So if you look up, if you Google trained immunity, you'll come up across something that reads a little bit like this, the long term changes seen in innate immune cell phenotypes. That's after priming with a pathogen product which leads to de facto induction of innate immune memory, now described as trained immunity. When somebody asks me in an elevator like what is trained immunity, I just give them the one liner, it's inducing innate immune memory against pathogens. That's what our laboratory is trying to do. So the products that we have tested before, we've described that these are equipotent. And why did we choose these products? Well, because they are synthetic. These are ultra pure synthetic clones of a vaccine adjuvant, a compound that we have all received already. It's been trained. It's called monophospholipid A. And so, however, the company that has the rights to that product will not develop it outside the use of a vaccine adjuvant. And so this company located in Alabama actually created clones of monophospholipid A. And I will tell you, these phosphorylated hexaacyl disaccharides, six chains, this one is the one that's only five chains in, which is why it's the most likely to be developed for human use. So how do these TLR4 agonists work? Well, they bind to the TLR4 protein and they lead to MIDI88 signaling, which influences cytokine production, and then also works through the TRIV pathway, which influences interferon factor three. Now let's talk about, I'm just going to have time to talk about two models that we've developed and used. So again, what I'm about to show you is independent of antibiotic therapy, fully recognizing that in a clinical patient, we would offer antibiotics. So we've already tested that these products work with systemic staphylococcus aureus and also abdominal sepsis, but we fully recognize that patients don't know when they're going to develop systemic infection or abdominal sepsis. But we do recognize with frailty indices and other comorbidities that there are some patients who are likely to develop pneumonia. And nosocomial pneumonia is associated with increased mortality, certainly higher in critically ill and elderly populations and associated with economic burden. And it is the highest cause of rehospitalization in sepsis survivors. Our laboratory works with these particular species because they're the most likely to develop resistance. So the model of pneumonia that we developed involves wild type 18 to 20 week old mice, where we administer intertracheal vehicle, which is just lactated ringers, 100 microliters or 20 micrograms of 3D fad mixed in 100 microliters. And so our treatment plan is at 48 hours and 24 hours before inoculation of klebsiella pneumoniae. It's a very virulent strain. You notice that there's just 2,300 bacteria that we administer. So we had four ongoing models. We have a model that we assess that did not get inoculated. And then we had, I'm sorry, a group. And then we had a group where we euthanized at six hours, one at 48 hours, and then one that we followed for survival up to 15 days. And what you observe here in the bronchial alveolar lavage fluid is that there is significant decline of bacterial burden at six hours. And this is sustained at 48 hours. However, we wanted to examine if there was a protection to the animal because sometimes you can clear the bacteria, but there may be no difference in survival. And so we here note that there was an 80% survival benefit for those animals treated with 3DFAD as compared to the vehicle-treated animals. We then wanted to examine if there are specific leukocytes that were responsible for enhancing clearance that may confer the survival benefit. So we looked at neutrophils, and these are the three groups here, no infection, a six-hour and 48-hour infection. And initially, what the product does when administered intratracheally, and we've observed this in the peritoneal cavity as well, is that there is a recruitment of neutrophils. Now in the lung, as you go from six hours, there's still an increase in neutrophils, but by 48 hours, it's matched. And we observed the same pattern with monocytes and with macrophages, which is that there is a rise initially, but once you introduce the inoculum, there seems to be no difference. We then wanted to examine what happens in the entire lung, because thus far, I've only been showing you bronchoalveolar lavage. So we treated the mice the same way at 48 and 24 hours before euthanasia. We took the entire lung tissue, homogenized it, and then we wanted to look for leukocytes. So neutrophils, monocytes, and in this case, it's interstitial macrophages, because only the alveolar macrophages are predominant in the alveolar space. They do have different biomarkers, so then these are interstitial. There are very, very few alveolar in the lung homogenate, which is why you don't see them reported here. But all of these seem to be higher in the treated animals, and also CD8 positive T cells were higher. We then wanted to examine if there are any antimicrobial properties that are enhanced with the treatment of 3DFED. And interestingly, in the bronchoalveolar lavage, there's actually higher phagocytosis in the vehicle. And by the way, all these results that I'm showing you are in triplicate, so we're fairly confident this is what it is. But when it comes to monocytes and macrophages, no difference. Now in the lung homogenate, we do see something different. We do see an increased number of phagocytosis in the neutrophils, monocytes, and macrophages. And the way we assess phagocytosis is we take this pH rotolabeled E. coli, and as this pH rotolabeled E. coli gets engulfed in the phagosome, the pH changes, and there is a fluorescence of red. And so we measure mean fluorescence intensity here, MFI. That's how we measure the phagocytosis. Let's talk about a different model now. So what our laboratory is discovering is that when we give these products, the cytokines are lower, and we know preserved organ function, but yet we have advanced antimicrobial functions. So we had a thought that maybe we're just regulating the inflammatory response system, and that may be a mechanism of why we're preserving organ function. So we took a model of ischemia, reperfusion-induced acute kidney injury, and we did that because cardiac and non-cardiac surgery patients have an incidence of AKI, especially in cardiac surgery, up to 40%, and when these patients develop a dependence on dialysis, their mortality is quite high, up to 70%. So our hypothesis was, you know, by inducing trained immunity, will we confer a regulated inflammatory response that will lead to a term used now is disease tolerance. You know, if the patient has a particular insult, will they be able to tolerate that? And so for this, we used a 3D6-asulfad, which is the other product we described there. And our treatment plan was here at four days before injury. We take blood. We give the animals a couple of days to recover from that, and then we treat just like we did before. I do want to point out that the dose here is 200 micrograms, which is 10 times higher than what we gave in the lung. Then on the day of surgery, under ketamine and xylosine anesthesia, we did provide bilateral renal artery clamping for 24 minutes. And then 24 hours later, we collect blood. And then three days after the injury, we collect blood and the organs. And what you can see here is that there's a significant preservation of organ function as assessed by BUN and creatinine at post-injury day one and three. This is a typo. This should be minus four. I apologize for that, because the baseline was taken four days before the injury. When we looked at biomarkers of injury, NGAL was significantly lower, which is typically in the distal tubules. Hemoxygenase 1 is not a marker of injury, but rather is expressed as a response to injury. And so this was also significantly lower. Interestingly, we did see an increase in interleukin-1 beta in the kidney tissue. We then wanted to look at what the effect was on the tissue of the kidney. And you see that the architecture of the tissue is preserved in the fat-treated animals, whereas you see casts here in the vehicle group. The histology plot shows the comparison of how fat-treated animals had a better preservation of the tissue. We then wanted to examine the expression of CHEM1, another biomarker of injury, which is typically in the proximal tubules. And so here in this immunofluorescent stain, CHEM1 is stained as red, so you can appreciate that there's less red here on the fat-treated animal kidney tissue. And the plot here of the outer medulla demonstrates that. I will say the cortex didn't have any differences between groups. So in summary, what we're aiming here is can we train the immune system to augment antimicrobial functions but yet have a regulated inflammatory response. This is important with a resistance to antibiotic therapy, which continues to rise. This could be a plausible approach to deal with this problem, and it can also lead to organ preservation, certainly in ischemia reperfusion-induced acute kidney injury, which is common in perioperative medicine. FADs appear to be strong candidates for pharmaceutical development, and in October of 2022, Revelation Pharmaceuticals entered an agreement with Vanderbilt to work and develop 3-DFAT to bring it to the bedside. So I don't know how long that will take, but that's starting now. Thank you for your attention.

microbial resistance

trained immunity

TLR4 agonists

pneumonia

acute kidney injury