Regulation of kynurenine metabolism by blood donor genetics and biology impacts red cell hemolysis in vitro and in vivo.

ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

Multiomics Signatures of Coagulopathy in a Polytrauma Swine Model Contrasted with Severe Multisystem Injured Patients.

Trauma-induced coagulopathy (TIC) is a leading contributor to preventable mortality in severely injured patients. Understanding the molecular drivers of TIC is an essential step in identifying novel therapeutics to reduce morbidity and mortality. This study investigated multiomics and viscoelastic responses to polytrauma using our novel swine model and compared these findings with severely injured patients. Molecular signatures of TIC were significantly associated with perturbed coagulation and inflammation systems as well as extensive hemolysis. These results were consistent with patterns observed in trauma patients who had multisystem injuries. Here, intervention using resuscitative endovascular balloon occlusion of the aorta following polytrauma in our swine model revealed distinct multiomics alterations as a function of placement location. Aortic balloon placement in zone-1 worsened ischemic damage and mitochondrial dysfunction, patterns that continued throughout the monitored time course. While placement in zone-III showed a beneficial effect on TIC, it showed an improvement in effective coagulation. Taken together, this study highlights the translational relevance of our polytrauma swine model for investigating therapeutic interventions to correct TIC in patients.

Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown that P. aeruginosa secretes outer-membrane vesicles that deliver a small RNA to human airway epithelial cells (AECs), in which it suppresses the innate immune response. Here, we demonstrate that interdomain communication through small RNA-containing membrane vesicles is bidirectional and that microRNAs (miRNAs) in extracellular vesicles (EVs) secreted by human AECs regulate protein expression, antibiotic sensitivity, and biofilm formation by P. aeruginosa Specifically, human EVs deliver miRNA let-7b-5p to P. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1-3, and increases the ability of beta-lactam antibiotics to reduce biofilm formation by targeting the beta-lactamase AmpC. Let-7b-5p is bioinformatically predicted to target not only PpkA, ClpV1, and AmpC in P. aeruginosa but also the corresponding orthologs in Burkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let-7b-5p to reduce biofilm formation and increase beta-lactam sensitivity is not limited to P. aeruginosa Here, we provide direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryote, resulting in subsequent phenotypic alterations in the prokaryote as a result of this interdomain communication. Since let-7-family miRNAs are in clinical trials to reduce inflammation and because chronic P. aeruginosa lung infections are associated with a hyperinflammatory state, treatment with let-7b-5p and a beta-lactam antibiotic in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

Omics Signatures of Tissue Injury and Hemorrhagic Shock in Swine.

OBJECTIVE: Advanced mass spectrometry methods were leveraged to analyze both proteomics and metabolomics signatures in plasma upon controlled tissue injury (TI) and hemorrhagic shock (HS)-isolated or combined-in a swine model, followed by correlation to viscoelastic measurements of coagulopathy via thrombelastography. BACKGROUND: TI and HS cause distinct molecular changes in plasma in both animal models and trauma patients. However, the contribution to coagulopathy of trauma, the leading cause of preventable mortality in this patient population remains unclear. The recent development of a swine model for isolated or combined TI+HS facilitated the current study. METHODS: Male swine (n=17) were randomized to either isolated or combined TI and HS. Coagulation status was analyzed by thrombelastography during the monitored time course. The plasma fractions of the blood draws (at baseline; end of shock; and at 30 minutes, 1, 2, and 4 hours after shock) were analyzed by mass spectrometry-based proteomics and metabolomics workflows. RESULTS: HS-isolated or combined with TI-caused the most severe omic alterations during the monitored time course. While isolated TI delayed the activation of coagulation cascades. Correlation to thrombelastography parameters of clot strength (maximum amplitude) and breakdown (LY30) revealed signatures of coagulopathy which were supported by analysis of gene ontology-enriched biological pathways. CONCLUSION: The current study provides a comprehensive characterization of proteomic and metabolomic alterations to combined or isolated TI and HS in a swine model and identifies early and late omics correlates to viscoelastic measurements in this system.

Omics markers of platelet transfusion in trauma patients.

BACKGROUND: Even in the era of the COVID-19 pandemic, trauma remains the global leading cause of mortality under the age of 49. Trauma-induced coagulopathy is a leading driver of early mortality in critically ill patients, and transfusion of platelet products is a life-saving intervention to restore hemostasis in the bleeding patient. However, despite extensive functional studies based on viscoelastic assays, limited information is available about the impact of platelet transfusion on the circulating molecular signatures in trauma patients receiving platelet transfusion. MATERIALS AND METHODS: To bridge this gap, we leveraged metabolomics and proteomics approaches to characterize longitudinal plasma samples (n = 118; up to 11 time points; total samples: 759) from trauma patients enrolled in the Control Of Major Bleeding After Trauma (COMBAT) study. Samples were collected in the field, in the emergency department (ED), and at intervals up to 168 h (7 days) post-hospitalization. Transfusion of platelet (PLT) products was performed (n = 30; total samples: 250) in the ED through 24 h post-hospitalization. Longitudinal plasma samples were subjected to mass spectrometry-based metabolomics and proteomics workflows. Multivariate analyses were performed to determine omics markers of transfusion of one, two, three, or more PLT transfusions. RESULTS: Higher levels of tranexamic acid (TXA), inflammatory proteins, carnitines, and polyamines were detected in patients requiring PLT transfusion. Correlation of PLT units with omics data suggested sicker patients required more units and partially overlap with the population requiring transfusion of packed red blood cell products. Furthermore, platelet activation was likely increased in the most severely injured patients. Fatty acid levels were significantly lower in PLT transfusion recipients (at time of maximal transfusion: Hour 4) compared with non-recipients, while carnitine levels were significantly higher. Fatty acid levels restore later in the time course (e.g., post-PLT transfusion). DISCUSSION: The present study provides the first multi-omics characterization of platelet transfusion efficacy in a clinically relevant cohort of trauma patients. Physiological alterations following transfusion were detected, highlighting the efficacy of mass spectrometry-based omics techniques to improve personalized transfusion medicine. More specialized clinical research studies focused on PLT transfusion, including organized pre and post transfusion sample collection and limitation to PLT products only, are required to fully understand subsequent metabolomic and proteomic alterations.

The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI.

Iron is an essential nutrient for plants, but excess iron is toxic due to its catalytic role in the formation of hydroxyl radicals. Thus, iron uptake is highly regulated and induced only under iron deficiency. The mechanisms of iron uptake in roots are well characterized, but less is known about how plants perceive iron deficiency. We show that a basic helix-loop-helix (bHLH) transcription factor Upstream Regulator of IRT1 (URI) acts as an essential part of the iron deficiency signaling pathway in Arabidopsis thaliana The uri mutant is defective in inducing Iron-Regulated Transporter1 (IRT1) and Ferric Reduction Oxidase2 (FRO2) and their transcriptional regulators FER-like iron deficiency-induced transcription factor (FIT) and bHLH38/39/100/101 in response to iron deficiency. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) reveals direct binding of URI to promoters of many iron-regulated genes, including bHLH38/39/100/101 but not FIT While URI transcript and protein are expressed regardless of iron status, a phosphorylated form of URI only accumulates under iron deficiency. Phosphorylated URI is subject to proteasome-dependent degradation during iron resupply, and turnover of phosphorylated URI is dependent on the E3 ligase BTS. The subgroup IVc bHLH transcription factors, which have previously been shown to regulate bHLH38/39/100/101, coimmunoprecipitate with URI mainly under Fe-deficient conditions, suggesting that it is the phosphorylated form of URI that is capable of forming heterodimers in vivo. We propose that the phosphorylated form of URI accumulates under Fe deficiency, forms heterodimers with subgroup IVc proteins, and induces transcription of bHLH38/39/100/101 These transcription factors in turn heterodimerize with FIT and drive the transcription of IRT1 and FRO2 to increase Fe uptake.

Omics Markers of Red Blood Cell Transfusion in Trauma.

Red blood cell (RBC) transfusion is a life-saving intervention for millions of trauma patients every year worldwide. While hemoglobin thresholds are clinically driving the need for RBC transfusion, limited information is available with respect to transfusion efficacy at the molecular level in clinically relevant cohorts. Here, we combined plasma metabolomic and proteomic measurements in longitudinal samples (n = 118; up to 13 time points; total samples: 690) from trauma patients enrolled in the control of major bleeding after trauma (COMBAT) study. Samples were collected in the emergency department and at continuous intervals up to 168 h (seven days) post-hospitalization. Statistical analyses were performed to determine omics correlate to transfusions of one, two, three, five, or more packed RBC units. While confounded by the concomitant transfusion of other blood components and other iatrogenic interventions (e.g., surgery), here we report that transfusion of one or more packed RBCs­mostly occurring within the first 4 h from hospitalization in this cohort­results in the increase in circulating levels of additive solution components (e.g., mannitol, phosphate) and decreases in the levels of circulating markers of hypoxia, such as lactate, carboxylic acids (e.g., succinate), sphingosine 1-phosphate, polyamines (especially spermidine), and hypoxanthine metabolites with potential roles in thromboinflammatory modulation after trauma. These correlations were the strongest in patients with the highest new injury severity scores (NISS > 25) and lowest base excess (BE < −10), and the effect observed was proportional to the number of units transfused. We thus show that transfusion of packed RBCs transiently increases the circulating levels of plasticizers­likely leaching from the blood units during refrigerated storage in the blood bank. Changes in the levels of arginine metabolites (especially citrulline to ornithine ratios) are indicative of an effect of transfusion on nitric oxide metabolism, which could potentially contribute to endothelial regulation. RBC transfusion was associated with changes in the circulating levels of coagulation factors, fibrinogen chains, and RBC-proteins. Changes in lysophospholipids and acyl-carnitines were observed upon transfusion, suggestive of an effect on the circulating lipidome­though cell-extrinsic/intrinsic effects and/or the contribution of other blood components cannot be disentangled. By showing a significant decrease in circulating markers of hypoxia, this study provides the first multi-omics characterization of RBC transfusion efficacy in a clinically relevant cohort of trauma patients.

Effect of leukoreduction on the metabolism of equine packed red blood cells during refrigerated storage.

BACKGROUND: Understanding of the biochemical and morphological lesions associated with storage of equine blood is limited. OBJECTIVE: To demonstrate the temporal sequences of lipid and metabolic profiles of equine fresh and stored (up to 42 days) and leukoreduced packed red blood cells (LR-pRBC) and non-leukoreduced packed RBC (nLR-pRBC). ANIMALS: Packed RBC units were obtained from 6 healthy blood donor horses enrolled in 2 blood banks. METHODS: Observational study. Whole blood was collected from each donor using transfusion bags with a LR filter. Leukoreduction pRBC and nLR-pRBC units were obtained and stored at 4°C for up 42 days. Sterile weekly sampling was performed from each unit for analyses. RESULTS: Red blood cells and supernatants progressively accumulated lactate products while high-energy phosphate compounds (adenosine triphosphate and 2,3-Diphosphoglycerate) declined. Hypoxanthine, xanthine, and free fatty acids accumulated in stored RBC and supernatants. These lesions were exacerbated in non-LR-pRBC. CONCLUSION AND CLINICAL IMPORTANCE: Leukoreduction has a beneficial effect on RBC energy and redox metabolism of equine pRBC and the onset and severity of the metabolic storage lesions RBC.

Trauma patients with type O blood exhibit unique multiomics signature with decreased lectin pathway of complement levels.

BACKGROUND: Patients with type O blood may have an increased risk of hemorrhagic complications because of lower baseline levels of von Willebrand factor and factor VIII, but the transition to a mortality difference in trauma is less clear. We hypothesized that type O trauma patients will have differential proteomic and metabolomic signatures in response to trauma beyond von Willebrand factor and factor VIII alone. METHODS: Patients meeting the highest level of trauma activation criteria were prospectively enrolled. Blood samples were collected upon arrival to the emergency department. Proteomic and metabolomic (multiomics) analyses of these samples were performed using liquid chromatography-mass spectrometry. Demographic, clinical, and multiomics data were compared between patients with type O blood versus all other patients. RESULTS: There were 288 patients with multiomics data; 146 (51%) had type O blood. Demographics, injury patterns, and initial vital signs and laboratory measurements were not different between groups. Type O patients had increased lengths of stay (7 vs. 6 days, p = 0.041) and a trend toward decreased mortality secondary to traumatic brain injury compared with other causes (traumatic brain injury, 44.4% vs. 87.5%; p = 0.055). Type O patients had decreased levels of mannose-binding lectin and mannose-binding lectin-associated serine proteases 1 and 2, which are required for the initiation of the lectin pathway of complement activation. Type O patients also had metabolite differences signifying energy metabolism and mitochondrial dysfunction. CONCLUSION: Blood type O patients have a unique multiomics signature, including decreased levels of proteins required to activate the lectin complement pathway. This may lead to overall decreased levels of complement activation and decreased systemic inflammation in the acute phase, possibly leading to a survival advantage, especially in traumatic brain injury. However, this may later impair healing. Future work will need to confirm these associations, and animal studies are needed to test therapeutic targets. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level IV.

Effect of leukoreduction on the omics phenotypes of canine packed red blood cells during refrigerated storage.

BACKGROUND: Red blood cell (RBC) storage promotes biochemical and morphological alterations, collectively referred to as storage lesions (SLs). Studies in humans have identified leukoreduction (LR) as a critical processing step that mitigates SLs. To date no study has evaluated the impact of LR on metabolic SLs in canine blood units using omics technologies. OBJECTIVE: Compare the lipid and metabolic profiles of canine packed RBC (pRBC) units as a function of LR in fresh and stored refrigerated (up to 42 days) units. ANIMALS: Packed RBC units were obtained from 8 donor dogs enrolled at 2 different Italian veterinary blood banks. STUDY DESIGN AND METHODS: Observational study. A volume of 450 mL of whole blood was collected using Citrate-Phosphate-Dextrose-Saline-Adenine-Glucose-Mannitol (CPD-SAGM) transfusion bags with a LR filter to produce 2 pRBC units for each donor, without (nLR-pRBC) and with (LR-pRBC) LR. Units were stored in the blood bank at 4 ± 2°C. Sterile weekly samples were obtained from each unit for omics analyses. RESULTS: A significant effect of LR on fresh and stored RBC metabolic phenotypes was observed. The nLR-pRBC were characterized by higher concentrations of free short and medium-chain fatty acids, carboxylic acids (pyruvate, lactate), and amino acids (arginine, cystine). The LR-pRBC had higher concentrations of glycolytic metabolites, high energy phosphate compounds (adenosine triphosphate [ATP]), and antioxidant metabolites (pentose phosphate, total glutathione). CONCLUSION AND CLINICAL IMPORTANCE: Leukoreduction decreases the metabolic SLs of canine pRBC by preserving energy metabolism and preventing oxidative lesions.

Pharmacological induction of chromatin remodeling drives chemosensitization in triple-negative breast cancer.

Targeted therapies have improved outcomes for certain cancer subtypes, but cytotoxic chemotherapy remains a mainstay for triple-negative breast cancer (TNBC). The epithelial-to-mesenchymal transition (EMT) is a developmental program co-opted by cancer cells that promotes metastasis and chemoresistance. There are no therapeutic strategies specifically targeting mesenchymal-like cancer cells. We report that the US Food and Drug Administration (FDA)-approved chemotherapeutic eribulin induces ZEB1-SWI/SNF-directed chromatin remodeling to reverse EMT that curtails the metastatic propensity of TNBC preclinical models. Eribulin induces mesenchymal-to-epithelial transition (MET) in primary TNBC in patients, but conventional chemotherapy does not. In the treatment-naive setting, but not after acquired resistance to other agents, eribulin sensitizes TNBC cells to subsequent treatment with other chemotherapeutics. These findings provide an epigenetic mechanism of action of eribulin, supporting its use early in the disease process for MET induction to prevent metastatic progression and chemoresistance. These findings warrant prospective clinical evaluation of the chemosensitizing effects of eribulin in the treatment-naive setting.

Cooperation of chromatin remodeling SWI/SNF complex and pioneer factor AP-1 shapes 3D enhancer landscapes.

The mechanism controlling the dynamic targeting of SWI/SNF has long been postulated to be coordinated by transcription factors (TFs), yet demonstrating a specific TF influence has proven difficult. Here we take a multi-omics approach to interrogate transient SWI/SNF interactors, chromatin targeting and the resulting three-dimensional epigenetic landscape. We utilize the labeling technique TurboID to map the SWI/SNF interactome and identify the activator protein-1 (AP-1) family members as critical interacting partners for SWI/SNF complexes. CUT&RUN profiling demonstrates SWI/SNF targeting enrichment at AP-1 bound loci, as well as SWI/SNF-AP-1 cooperation in chromatin targeting. HiChIP reveals AP-1-SWI/SNF-dependent restructuring of the three-dimensional promoter-enhancer architecture and generation of enhancer hubs. Through interrogation of the SWI/SNF-AP-1 interaction, we demonstrate an SWI/SNF dependency on AP-1-mediated chromatin localization. We propose that pioneer factors, such as AP-1, bind and target SWI/SNF to inactive chromatin, where it restructures the genomic landscape into an active state through epigenetic rewiring spanning multiple dimensions.

Metabolic correlates to critical speed in murine models of sickle cell disease.

Introduction: Exercise intolerance is a common clinical manifestation in patients with sickle cell disease (SCD), though the mechanisms are incompletely understood. Methods: Here we leverage a murine mouse model of sickle cell disease, the Berkeley mouse, to characterize response to exercise via determination of critical speed (CS), a functional measurement of mouse running speed upon exerting to exhaustion. Results: Upon observing a wide distribution in critical speed phenotypes, we systematically determined metabolic aberrations in plasma and organs-including heart, kidney, liver, lung, and spleen-from mice ranked based on critical speed performances (top vs. bottom 25%). Results indicated clear signatures of systemic and organ-specific alterations in carboxylic acids, sphingosine 1-phosphate and acylcarnitine metabolism. Metabolites in these pathways showed significant correlations with critical speed across all matrices. Findings from murine models were thus further validated in 433 sickle cell disease patients (SS genotype). Metabolomics analyses of plasma from 281 subjects in this cohort (with HbA < 10% to decrease confounding effects of recent transfusion events) were used to identify metabolic correlates to sub-maximal exercise test performances, as measure by 6 min walking test in this clinical cohort. Results confirmed strong correlation between test performances and dysregulated levels of circulating carboxylic acids (especially succinate) and sphingosine 1-phosphate. Discussion: We identified novel circulating metabolic markers of exercise intolerance in mouse models of sickle cell disease and sickle cell patients.

Trans-Omics analysis of post injury thrombo-inflammation identifies endotypes and trajectories in trauma patients.

Understanding and managing the complexity of trauma-induced thrombo-inflammation necessitates an innovative, data-driven approach. This study leveraged a trans-omics analysis of longitudinal samples from trauma patients to illuminate molecular endotypes and trajectories that underpin patient outcomes, transcending traditional demographic and physiological characterizations. We hypothesize that trans-omics profiling reveals underlying clinical differences in severely injured patients that may present with similar clinical characteristics but ultimately have very different responses to treatment and clinical outcomes. Here we used proteomics and metabolomics to profile 759 of longitudinal plasma samples from 118 patients at 11 time points and 97 control subjects. Results were used to define distinct patient states through data reduction techniques. The patient groups were stratified based on their shock severity and injury severity score, revealing a spectrum of responses to trauma and treatment that are fundamentally tied to their unique underlying biology. Ensemble models were then employed, demonstrating the predictive power of these molecular signatures with area under the receiver operating curves of 80 to 94% for key outcomes such as INR, ICU-free days, ventilator-free days, acute lung injury, massive transfusion, and death. The molecularly defined endotypes and trajectories provide an unprecedented lens to understand and potentially guide trauma patient management, opening a path towards precision medicine. This strategy presents a transformative framework that aligns with our understanding that trauma patients, despite similar clinical presentations, might harbor vastly different biological responses and outcomes.