Mechanisms of Immune Dysfunction after Trauma and Surgical Sepsis

Project Summary/Abstract Trauma and Surgical Sepsis are among the leading causes of morbidity and death worldwide. Both of these acute insults can lead to immune dysfunction that then contributes to a state of persistent critical illness. This immune dysfunction is manifested by an excessive systemic inflammatory response that can lead to organ dysfunction; and a simultaneous suppression of immune defenses that renders patients susceptible to secondary infections. However, we lack a comprehensive and integrated view of how humans respond to severe injury, and more importantly, how these responses differ between patients that recover quickly vs. those that die and/or development persistent critical illness. Advances in single cell multiomics and systemic multi-platform, multiomics now makes it possible to characterize changes across a broad range of cell states and patterns with the circulating biomolecule landscape to great depth. In the previous funding cycle, we were the first to apply single cell genomics and large-scale multi-platform, multiomics of blood samples in severely injured patients. This published work identified many novel findings, including the early massive release of cellular constituents in trauma patients that follow a complicated course or die. In addition, open chromatin analysis of PBMC found that patients who remain critically ill also have global epigenetic changes evident early across immune cell types, representing de-repression of polycomb targets. In one line of research, we will reverse translate the these and other dramatic findings from our initial human multiomic analyses into our mouse model of hemorrhagic shock and trauma to pursue potential therapeutic targets. In another line of research, we will extend our multiomic analysis to create a Blood Atlas of the human trauma response. This online resource will incorporate data on the range of circulating immune cell states commonly seen after severe injury and integrate these with the longitudinal changes in high dimensional datasets of circulating proteins, lipids and other metabolites. We will apply state-of-the-art computational strategies to identify biomarkers and therapeutic targets with patients stratified by outcomes, treatments, age and sex. We will compare our results with similar published studies in sepsis. In addition to the discoveries and mechanistic insights our analysis will yield, we hope that the resources we provide will stimulate comparative studies and further analyses of our datasets. Research Strategy Scientific Justification Hemorrhage due to trauma-induced coagulopathy (TIC) is a key driver of mortality in severely- injured patients. Coagulation phenotypes are dynamic post-injury, rapidly transitioning from an anti-coagulant to pro-coagulant state (1). These states are highly dependent on a variety of extrinsic variables such as dilution of blood with resuscitation fluids, environmental temperature, and acidosis (2). The complex nature of the coagulation phenotype post-injury may be better elucidated in a well-controlled animal model and we believe the studies outlined below will provide important data regarding the role of cell death mechanisms in driving coagulation phenotypes. Our extensive matrix of animal modeling studies provide an excellent opportunity to characterize the role of these processes in driving TIC. The proposed equipment purchase of a ROTEM delta (Werfen. Bedford, MA) will be used to generate coagulation profiles of mouse plasma obtained using our hemorrhagic shock model. We have proposed to use this animal model extensively in the parent R35, as it is the central research which comprises Strategy 3, summarized below. Acquisition of this additional data from plasma already in hand will bolster our already-extensive multi-omic analyses and move our research in a novel direction by filling key gaps in our characterization of trauma phenotypes. Adding a coagulation “-omics” component to our existing multi-omic workflow will allow for mechanistic and correlation analysis not previously performed in trauma. Strategy 3. Use of multi-platform, multi-omics to characterize the systemic response to injury We will subject mice to severe (3 hour) and mild, resolving (1 hour) hemorrhagic shock according to our standard model. Blood will be withdrawn at the appropriate endpoint using cardiac puncture and divided into a heparinized tube for proteomic/metabolomic analysis and a citrated tube for ROTEM analysis. Of note, studies will be performed on both male and female mice in order to define the key differences in traumatic phenotypes associated with sex. The treatment groups currently in progress or undergoing breeding crosses are outlined in the chart below. Animal model Pathway targeted male female mild severe WT mice + necrostatin Necroptosis x x x x WT mice + ferrostatin Ferroptosis x x x x WT mice + Z-vad Apoptosis x x x x PAD4-/- mice Netosis x x x x Gasdermin D-/- mice Pyroptosis x x x x Gasdermin D (fl/fl); LysM-Cre+ mice Pyroptosis in myeloid cells x x x x Gasdermin D (fl/fl); Alb-Cre+ mice Pyroptosis in liver cells x x x x Gasdermin D (fl/fl); VE-Cre+ mice Pyroptosis in endothelial cells x x x x Gasdermin D (fl/fl); alphaMHC-Cre+ mice Pyroptosis in cardiomyocytes x x x x Gasdermin D (fl/fl); PF4-Cre+ mice Pyroptosis in megakaryocyte lineage cells x x x x Ninj1-/- mice Pyroptosis x x x x Ninj1(fl/fl); LysM- Cre+ mice Pyroptosis in myeloid cells x x x x Ninj1(fl/fl); Alb-Cre+ mice Pyroptosis in liver cells x x x x Ninj1(fl/fl); VE-Cre+ mice Pyroptosis in endothelial cells x x x x Ninj1(fl/fl); alphaMHC-Cre+ mice Pyroptosis in cardiomyocytes x x x x Ninj1 (fl/fl); PF4- Cre+ mice Pyroptosis in megakaryocyte lineage cells x x x x Original readouts proposed for mouse plasma: ALT/AST (Heska); metabolomic profiles (Metabolon, Inc, Morrisville, NC); proteomic profiles (Creative Proteomics, Shirley, NY) New data that can be added with proposed equipment: clotting time, clot formation time, maximum clot firmness, alpha-angle, lysis index, and maximum lysis Plans for future recurring costs The requested instrument will require a service contract and consumable supplies. The Billiar laboratory has access to other funding sources as well as robust Departmental support which will guarantee coverage of the costs for service contracts as well as consumables for the term of the parent R35 award. Plans for equipment training and upkeep The Department of Surgery research laboratories employ key technical staff members who are responsible for instrument set-up, managing service contracts, and for performing weekly maintenance checks. These technicians are trained by the company representatives and will then train and new staff members who require use of the equipment. Product manuals and technical representative contact information is kept with the instrument and available to all users. References 1. Klienveld DJ, Hamada SR, Sandroni C. Trauma-induced Coagulopathy. Intensive Care Medicine; 2022 Nov;48(11):1642-1645. 2. White NJ. Mechanisms of Trauma-induced Coagulopathy. Hematology Am Soc Hematol Educ Program; 2013 (1): 660-663.