Good afternoon, everybody. I would like to thank the organizers first to give me the opportunity to present our recent research work on how ethanol represses LC3-associated phagocytosis in human macrophages via sirtuin 2. As we all know that sepsis is a dysregulated host immune response to infection. Sepsis and septic shock together kill more than 270,000 people in the U.S. alone, and alcohol abuse is reported in one in eight critically ill patients. It increases the mortality and suppresses immune response to sepsis. It is an independent risk factor for death in sepsis patients. Sepsis, as we all know, it has different stages. It first starts with the hyper-inflammatory phase, which is then progresses, transitions gradually to the hypo-inflammatory phase. The hyper-inflammatory phase is characterized by active phagocytosis of the pathogens by the immune cells and higher glycolysis, which is gradually going to the hypo-inflammatory phase, which is characterized by dysregulated phagocytosis. Ethanol or alcohol is known to dysregulate phagocytosis and it shortens the hyper-inflammatory phase. This talk today will only focus on the effect of ethanol on the hyper-inflammatory phase of sepsis, and we will only focus on the immune cell functions, particularly macrophages. Previous work from our laboratory have shown that ethanol impairs bacterial clearance in mice. Here on the left side graph, if you'll see that in the control versus the sepsis, when the mice were exposed to sepsis, like mice were intraperitoneally injected with the sickle slurry, and that is the mouse sepsis model we use here, and then we see the bacterial load, which is in the y-axis, is going up. In presence of ethanol, where mouse were fed with ethanol in the drinking water for 11 days, and then we see the bacterial load is higher in the peritoneal lavage. Seven days post-sepsis, while in the vehicle-treated mice and the septic mice from the normal wild-type mice where bacteria were cleared in the peritoneal lavage, but in the mice which were previously exposed with ethanol, they could not clear the bacteria. That means we can come to the conclusion that ethanol impairs bacterial clearance in mice. Glycolysis plays a critical role in phagocytosis because glycolysis ultimately produces lactate and that reduces the pH and that facilitates phagocytosis. Glycolysis is a multi-enzyme-mediated process in which glucose is taken up by glucose transporters and then gradually converted to pyruvate through other metabolites. There are several steps in glycolysis. Three of them are called the regulatory phases or the enzymes. Since you can see in this cartoon there are like double bonds, like it's a reversible reaction, only three of them, there are single way the arrow is indicating. That means they are the critical steps. They are the hexokinase, hexokinase 1 and 2, then phosphofructokinase. Phosphofructokinase has three isozymes, PFKP, PFKL, and the PFKM. PFKP is the platelet isoform. And the third is the pyruvate kinase. And glycolysis not only fuels the immune cells during inflammation, it also facilitates bacterial clearance through phagocytosis. There is a newer form of phagocytosis, or we should call it a separate form of phagocytosis, which is called LC3-associated phagocytosis, which, what is the difference? Because it is a quicker form of phagocytosis when people get infected or mice get infected or cells get infected, they need to clear the bacteria as fast as possible. Compared to the classical phagocytic routes, this is a much faster route of phagocytosis. In this process, this cartoon shows the apoptotic cells of the phagocytic cargo is engulfed by the phagocytic cells, maybe macrophage or dendritic cells, and then they are converted to a laposome through incorporation of the LC3B into the LC3-2 on the membrane of these laposomes. So what happens? LC3 gets broken down to LC3-1 and 2, which is shown here as LC1 and LC2, and that gets incorporated on the membrane of those lapophores. PFKP, the glycolytic enzyme, has already been shown to play a part in the breaking down of LC1 to LC2 by phosphorylating a protein called ATG4B. There is another talk tomorrow. I will go in detail on how PFKP interacts with these things later on. So in short, PFKP regulates activation of LC, then the laposomes are formed, laposomes clear bacteria. SIRTWIN2 is known to modulate both phagocytosis and glycolysis. In the SIRTWIN2-deficient mice, we have seen protection in sickle cell induced sepsis as well as in staph aureus infection by other people, and SIRTWIN2 inhibition also modulates the metabolic fitness of immune cells through the regulation of glycolytic enzyme PFKP, particularly in the T cells. Based on this previous literature and our work, we have come to this working hypothesis that SIRTWIN2 impairs phagocytosis through PFKP following acute ethanol exposure in the human macrophages. To test our hypothesis, we have received whole blood from the healthy volunteers, then using the magnetic beads, we have isolated the monocytes. They were differentiated for six days in presence of MCSF and the macrophage colony stimulating factor, and then these differentiated macrophages were either treated to like PBS as the vehicle or the ethanol, 25 micromolar for 20 hours, and for the last four hours, either they got PBS or LPS as the cellular model of sepsis. First, we looked for SIRTWIN2 expression by two methods. One is the immunocytochemistry. As you can see here, the green expression, that is the SIRTWIN2, and the blue is the DAPI. The green expression, you can see, is going higher following ethanol exposure in presence of LPS, and also we have confirmed this immunocytochemistry by Western blot techniques. And then we looked for whether ethanol mutes phagocytosis or not. To do that, we have used a bioparticle called the P-H rotozyme A. P-H rotozyme A is known to be red in acidic media. So what happens, this is like previously people used to use other beads, but these beads are colorless. When the cells engulf them and then they merge with the phagosomes or phagophores are formed, and the P-H inside the phagophore is acidic, that's why they become red, and that's why we see these red colors. We can see after LPS challenge, there's more phagocytosis as we see more red beads inside the cells. But if the cells were exposed to ethanol, you don't see that many red beads, and these were quantified, and we have found that ethanol impairs phagocytosis. If SIRTWIN2 is a major player in phagocytosis, inhibition of SIRTWIN2 should reverse this process, this inhibition, and in the inhibitor-exposed cells, then phagocytosis should increase. Yes, indeed, we have seen here when the cells were exposed with the SIRTWIN2 inhibitor, AK7, then there is increased phagocytosis, which confirms that SIRTWIN2 impairs phagocytosis during ethanol exposure. Then we next look for the glycolytic enzyme PFKP, what I was telling you before. We have also looked for the other isoforms of phosphofructokinase, PFKL, and M, in the mouse BMDMs also, and we did not see any change. That's why we were concentrating on PFKP. And here we can see that in this human macrophages, PFKP expression is going down following ethanol exposure in presence or absence of LPS, and then when the SIRTWIN inhibitor was added, the AK7, that preserves PFKP expression following ethanol exposure. Then now we know that SIRTWIN is modulating phagocytosis, SIRTWIN is modulating PFKP expression, then what happens to the LC3 activation? Then we have found that LC3 activation is going down in ethanol-exposed cells. If you see in the second bar, the LC32 expression, and in presence of the AK7, the SIRTWIN inhibitor, we have seen increase in LC3 1 and 2 expression. That shows that if we inhibit SIRTWIN2, then LC3 activation is going high, and LC3, as we remember, it is needed for the LC3-associated phagocytosis or LAP. Then next we looked for LAP formation. So you can see here the green color is the LC3, and the red beads inside the green color are the pH rodose. So that shows that LAP formation. And we have seen here that acute ethanol exposure mutes LAP formation, and you can see following the ethanol-exposed, in the ethanol-exposed cells, plus minus LPS, there is red and green, these dots are less. So if it is SIRTWIN2-mediated process, then again, inhibition of SIRTWIN2 should reverse the process, yes. We have seen more LAP or laposome formation when the cells were exposed to AK7. So from this data, we can conclude that ethanol, in presence or absence of LPS, it is increasing the SIRTWIN2, the only cytosolic SIRTWIN. That decreases PFKP, that decreases LC3 activation. Consequently, that decreased the LAP formation. And when we reversed, or when we prevented the SIRTWIN2 activity by its potent inhibitor AK7, then we see reversal of PFKP, LC3 activation, and LAP. And at the end, I would like to thank my mentor, Dr. Vidula Vachirajani, for her encouragement and support, and I would also like to thank my collaborators, Dr. Rachel Schrager and Dr. Laura Nagy, and my lab members, and the last but not the least, the NIH for their financial support. Thank you very much. I'm open to any questions. Thank you. I think we'll take the questions at the end so that we don't go overboard on our time. So I think I'll call our next speaker, Joaquin. Joaquin. All right, come on in. Good afternoon, everybody. My name is Joaquin Cantos. I'm an ICU physician and Intensive Care Research Fellow at the Italian Hospital of Buenos Aires in Argentina. And today I'm gonna be presenting our study named Allactic Base Excesses in an Independent Predictor of Death in Sepsis, a Propensity Score Analysis. The learning objectives of today's presentation are the following. In first place, negative allactic base excess is an independent predictor of in-hospital mortality in septic patients, even in those with preserved glomerular function. Acid-base disturbances reflected by allactic base excess could reveal tubular function impairment in the absence of established kidney damage in critically ill septic patients. And finally, the degree of acidemia in critically ill septic patients with hyperlactatemia is closely related to kidney function. Our study's rationale was based on two main concepts. One of them was allactic base excess. Allactic base excess is a novel concept introduced by Dr. Gattinoni in 2019, which quantifies the role of renal function in the acid-base balance of septic patients. It is calculated by the sum of standard base excess and lactate. It pays special attention to the role of fixed acids other than lactate in the plasma of septic patients. Particularly negative lactic base excess values represent the elevation of nonvolatile fixed acids other than lactate in plasma of these patients, noting the kidney function impairment. The other concept was sepsis-associated kidney injury, acute kidney injury, sorry. As it has been widely studied, this syndrome's pathophysiology involves multiple mechanisms. However, its diagnosis is still based on elevated serum creatinine and or decreased urine output. With the association of sepsis diagnosis. In this context, one of the limitations of this definition is that there has been found the dissociation between structural or functional changes of the kidney during this syndrome. Especially the role of tubular dysfunction, which has led to numerous investigations since 2012 in which they found and named this new syndrome called subclinical kidney injury. In particular, in the recent years, a lot of studies have been made concerning noble acute kidney injury biomarkers. These biomarkers are found to be present in situations of stress or damage previous to elevation of serum creatinine or impairment of glomerular function markers. In this context, the AdKey group presented a recent consensus statement in 2020 in which the proposed widened acute kidney injury definition in which they included these AdKey biomarkers into the definition of acute kidney injury, giving the syndrome of subclinical AdKey a greater presence in this definition. Given this background, we thought about the hypothesis that a negative lactic base excess in patients with preserved glomerular function or with no acute kidney injury criteria could represent this syndrome of subclinical AdKey or of tubular dysfunction. However, if this was true, this finding should be negative lactic base excess in patients with no acute kidney injury should be related to poor outcome. Therefore, our objectives were to evaluate if acid-base disturbances reflected by a lactic base excess could be predictive of unfavorable outcomes in septic patients independently of kidney injury. Also to evaluate whether a lactic base excess changes in septic patients behaved as predictors of in-hospital mortality even in those with glomerular filtration rate greater than 60. Lastly, we evaluated whether lactic acidosis was associated with acidemia in septic patients with and without acute kidney injury. To attain these objectives, we carried out a retrospective cohort study in two medical surgical ICUs in two teaching hospitals in Buenos Aires, Argentina. Our inclusion period was between January 2015 and January 2020. We included all patients without a diagnosis of sepsis or septic shock at ICU admission and we excluded those with kidney replacement therapy and or kidney transplant history at ICU admission as well as patients without data to calculate the lactic base excess or the SOFA score. After inclusion, the patients were divided in three groups. Negative lactic base excess, neutral lactic base excess, and positive lactic base excess. Patients were followed up until hospital discharge or in-hospital death. Our primary outcome was in-hospital mortality related to a lactic base excess groups adjusting by acute kidney injury and other confounders. We set two secondary outcomes. In-hospital mortality associated with a lactic base excess groups in patients with glomerular infiltration rate greater than 60 and the association between lactic acidosis and acidemia in septic patients with and without acute kidney injury. Regarding data analysis, we performed a Cox regression to evaluate the hazard ratio of mortality in the different lactic base excess groups. And we performed a propensity score adjustment by inverse probability weighting to control the confounding factors as our population was very heterogeneous. And we performed also a sensitivity analysis with adjusted Cox regression with the same confounding factors used in the propensity score. Starting with the results section, I present here our inclusion and exclusion criteria flowchart, 1,790 patients with a clinical suspicion of infection or a documented infection without history of kidney replacement therapy or kidney transplant were admitted to the ICU. We excluded 612 patients and finally we included 1,178 patients for follow-up. Afterwards, the division was made and we found that 53% of the population had neutral lactic base excess, 22% had negative lactic base excess, and 24% presented positive lactic base excess. Here I present a summarized table of the most relevant demographic data on our population. And what I would like to highlight is that negative lactic base excess patients presented higher Apache, SOFA, and Charlson scores as well as greater proportions of septic shock, mechanical ventilation requirement, and ICU length of stay. Regarding the clinical and lactic base data, this group of patients also presented the worst results, lower pH values, greater proportions of metabolic acidosis, and higher values of chloride-corrected unmeasured anions concentrations in plasma. Finally, I summarize here the most relevant laboratory and clinical data regarding kidney function. And we found that this group of patients with negative lactic base excess presented higher plasma creatinine values, lower glomerular filtration rates, higher proportions of chronic kidney disease, as well as acute kidney injury, and higher proportions of kidney replacement therapy requirements. Regarding our primary outcome, negative lactic base excess was found to be an independent predictor of in-hospital mortality in this population, whereas no difference was found between neutral and positive patients. After performing our sensitivity analysis, we also found this difference to be statistically significant, and no difference was found between neutral and positive lactic base excess group of patients. Regarding our secondary outcomes, patients with glomerular filtration rate greater than 60 and negative lactic base excess had higher in-hospital mortality than patients with neutral lactic base excess in the multivariate analysis. On the other hand, patients with glomerular filtration rate greater than 60 and positive lactic base excess had the same in-hospital mortality as patients with neutral lactic base excess, both in the univariate and multivariate analysis. Our other secondary outcome was the association between lactic acidosis and acidemia in patients with and without acute kidney injury. We found that patients with lactic levels between 2.5 and 5.6 had different pH values depending on the presence of acute kidney injury. This result was found to be both clinically and statistically significant. On the other hand, when lactic levels were higher than 5.6, pH values were equal in both groups, and both groups presented acidemia, both no acute kidney injury and acute kidney injury patients. The main finding of our study was that negative lactic base excess was an independent risk factor for mortality in clinically ill septic patients, even in those with preserved glomerular function. Additionally, there are some findings that are worth discussing. One of them is that we found that this group of patients presented a mean glomerular filtration rate of 26, which we thought it was higher than the theoretical value that should induce fixed acid retention that we thought about it should be or is around 15 milliliters per minute or less. Also, we found, as said before, a higher proportion of high anion gap metabolic acidosis as well as elevated unmeasured anions in this group of patients. This was a particularly interesting finding, mainly in those patients with preserved glomerular function or no acute kidney injury criteria, in which we consider that due to the reduced tubular acid secretion, urinary acidification was altered. So in the context of metabolic acidosis, this compensatory mechanism was not working well. Additionally, I would like to mention another interesting finding that 46% of patients with negative lactic base excess had CKD. As this is a risk factor for mortality, for acute kidney injury, and for tubular function impairment, we consider this variable as a confounding factor. However, we found the relationship between mortality and negative lactic base excess was independent of the presence of this disease. I present here some conclusions and future directions of our research based on our findings. Given that negative lactic base excess reflects acid-base disturbances in septic with preserved glomerular function, we consider lactic base excess as a potential marker of tubular dysfunction. Moreover, we would expect that in longitudinal studies or prospective studies to find altered urinary acidification in the urinary biochemistry. Another important issue is that we consider or we conceive that dynamic lactic base excess monitoring could be a possibility to detect progression to acute kidney injury. In particular, we think that patients with negative lactic base excess, this means with fixed acid retention and no acute kidney injury criteria or no signs of glomerular function impairment, that not normalized negative lactic base excess values could progress to acute kidney injury. And finally, we also think that given that novel acute kidney injury biomarkers are expensive and not readily available in all centers, particularly in our country, we consider a lactic base excess monitoring as an interesting alternative to detect the syndrome of subclinical acute kidney injury, given that it's a widespread resource and it could be obtained from a blood sample. And also, we consider that it could be also complementary to novel acute kidney injury biomarkers to give the name to the subclinical acute kidney injury, just because we consider it could give additional information. Our study had the following strengths that were that we included a large septic population, we defined acute kidney injury with K-DIGO criteria, and we included data regarding chronic kidney disease in our population. On the side of our limitations, we used cut-off values for building the lactic base excess groups. We had no information about history of diuretic therapy in our patients, and neither we did have data on nephrotoxic agents such as antibiotics, and we had some CKD missing data, however, we achieved our minimal sample size calculated. No disclosures to present. This was the bibliography used to build this presentation, and thank you very much.

lactic base excess

septic patients

acid-base disturbances

kidney function

tubular dysfunction

in-hospital mortality