Thank you, Dr. Oktober. So for those of you that I don't know, I'm Michael Ripple. I'm a fellow in pediatric critical care medicine at Emory University in Children's Health Care of Atlanta. I'm going to be talking today about one of our recently published projects on expression patterns of airway fluid cytokines in kids that have been intubated with pediatric ARDS. As far as disclosures, just our funding for some of the authors that were on the project. So one of the interests of our lab and our group is in the adaptive immune response, in particular the T-cell response in pediatric ARDS. There have been a few numerous studies that have kind of looked at this and touched on this. One study that demonstrated this CD4 to CD8 to CD4 T-cell ratio associated with disease severity in kids that have been admitted to the hospital for viral respiratory tract infections. Probably one of the best most studied or most described pathways or cell types is the Th17 cells or T helper 17 cells that affect pediatric ARDS severity through neutrophil recruitment to the airway and the alveolus and by affecting alveolar permeability. And in addition, Th17 cells are kind of a pro-inflammatory cell type. They act in balance or in concert with regulatory T-cells or Tregs. And one study in adults with ARDS demonstrated that the Th17 cell to Treg cell ratio was associated with or predicted mortality in adults. Numerous studies recently have started to look at or have used cytokines as biomarkers with various studies on pediatric ARDS demonstrating that IL-6, 8, 10, 18, MIP-1 beta and soluble TNF receptor 1 and 2 have been associated with various outcome measures, including ARDS severity, ventilator free days, non-pulmonary organ dysfunction and mortality. And while these studies are fantastic for prognostication and research on endotyping of ARDS, they may not completely recapitulate what's happening at the level of the alveolus or the airway. And so one of our interests was to kind of better describe those immune processes in order to, potentially in the future, test, you know, altering those pathways to affect these outcomes. So to accomplish this, we designed a study where we wanted to look at T-cell cytokines. So we used a commercially available 21 plex high sensitivity T-cell array and measured levels of those 21 cytokines on in the airway fluid of patients with ARDS. And then we also looked at a couple of pretty well-described serum biomarkers for pediatric ARDS, soluble TNF receptor 1 and RAGE. And so the patients that we included or enrolled in this study, all of them had pediatric ARDS per PALICC 1 criteria. They were stratified by severity into severe and not severe patients based on their OI or OSI on the day of sample collection. They were all children, obviously. And then we collected airway fluid samples through endotracheal, like routine endotracheal tube suctioning within 72 hours of intubation for respiratory failure due to ARDS. So as for the patients that were in the study, we had 47 patients in total. 34 of them fell into the non-severe, so like mild or moderate pediatric ARDS and then 13 in the severe group, not surprisingly because they were stratified based on disease severity. They were significantly different between numerous markers of kind of disease severity, including PILOD hospital days, VICU days, vent days, and the need for ECMO support. But there were no differences between the two groups in any of the demographic categories or time to sample collection. So for the analysis, like I said, we had 23 total analytes. Two of them were excluded prior to analysis. IL-2 and GM-CSF each had like 40 and 60% approximately of their values below the lower limit of detection for the assay. So we didn't look at those which left us with 21 analytes, 21 cytokines that we compared in severe and non-severe pediatric ARDS patients. Nine of them were actually significantly different between the two groups, the majority of which I'll talk about on the subsequent slides. 12 of them were not different and they're listed here on this slide on the right. The pathways that the significantly different cytokines kind of fell into were not surprisingly didn't, it was not just a single pathway or single cell type or signaling a pathway that was different between the two groups. There were cytokines that are involved in effector cell and Th1 development and activation, Th2 development, cytokines involved in the innate immune response, then also thankfully and not surprisingly Th17 mediators. So starting with the Th17 signaling pathways, so IL-17A and IL-21 were both significantly elevated in patients with severe pediatric ARDS. With IL-17A having probably the biggest increase between the two groups, about a five-fold increase in severe patients. As I said, these IL-17A affects alveolar neutrophil migration into the alveolus, permeability of fluid, organ dysfunction, et cetera. And there is that association with Th17 and Treg cell levels and their association with mortality. Also noteworthy is IL-21, which is required for Th17 cell differentiation from naive T cells and required for the production of IL-17 by Th17 cells. As far as the innate immune response, IL-8 popped up also, which is again, kind of not surprisingly, mostly because it's been used in a couple different studies in a couple different models in predicting pediatric ARDS severity and mortality. As far as its mechanism, it's a neutrophil activator and chemoattractant, and like I said, it has been demonstrated to be a predictor of worse outcomes in patients with pediatric ARDS in a couple different models. FractalKind has not been well studied in ARDS from what I could tell, but it is a cytokine or a chemokine that is expressed by monocytes, NK cells, T cells, smooth muscle cells, et cetera, and affects the migration and differentiation of those cell types. And then the other kind of group of cytokines that popped up that was interesting because it hasn't been well described in ARDS or pediatric ARDS is the markers of Th2 activation, so IL-4, IL-5, and IL-13. These are like three of the really big kind of canonical type two or Th2 inflammatory cytokines, all of which were significantly elevated in our patients with severe disease. One of the roles that has been demonstrated in adults with ARDS is the promotion, the repolarization of pro-inflammatory M1 macrophages, which are more active early in ARDS course to anti-inflammatory like restorative or reparative M2 macrophages, which when present later in the disease course in adults with ARDS, those adults do better. But there's been no description or no role yet that I've seen that for what the role of Th2 cells or this group of cytokines could be doing kind of early in the disease course, which is what we're sampling here. So conclusions from our small study, patients with severe pediatric ARDS do demonstrate a different profile, not surprisingly, of inflammatory cytokines and chemokines than patients with non-severe disease. And the pathways that are different, or there are multiple different pathways that are kind of different or upregulated in severe disease, demonstrating kind of roles for both innate immune cell pathways and T cells, and giving us a few different targets to kind of look at in future studies. So some of the things that our group will be doing in the future is certainly better describing the role of, and getting into some mechanistic work on the role of Th17 cells, specifically in pediatric ARDS, because a lot of the work has been done in adults. One of the limitations of this study was that this was a single snapshot in time of airway fluid cytokines. So we are definitely planning on kind of collecting more samples over time to see how the cytokine profiles change, and how they might change as disease severity changes, so relating it to oxygenation index. And then also certainly correlating it with some of the serum cytokines and biomarkers that have been previously described. We'll be doing that in the near future as well. So acknowledgments, definitely I'd like to thank my mentors, Dr. Fitzpatrick and Dr. Grunwell, as well as Dr. Stevenson and Ahmad, who are our two, our lab manager and lab tech who were responsible for processing all the human samples, patient samples, and for keeping the lab running when we're on busy clinical weeks. This is the reference for the paper that has been recently published on this work. I'm happy to take any questions.

Dr. Michael Ripple

expression patterns

airway fluid cytokines

pediatric acute respiratory distress syndrome

immune processes