Immune and metabolic markers for identifying and investigating severe Coronavirus disease and Sepsis in children and young people (pSeP/COVID ChYP study): protocol for a prospective cohort study.

INTRODUCTION: Early recognition and appropriate management of paediatric sepsis are known to improve outcomes. A previous system's biology investigation of the systemic immune response in neonates to sepsis identified immune and metabolic markers that showed high accuracy for detecting bacterial infection. Further gene expression markers have also been reported previously in the paediatric age group for discriminating sepsis from control cases. More recently, specific gene signatures were identified to discriminate between COVID-19 and its associated inflammatory sequelae. Through the current prospective cohort study, we aim to evaluate immune and metabolic blood markers which discriminate between sepses (including COVID-19) from other acute illnesses in critically unwell children and young persons, up to 18 years of age. METHODS AND ANALYSIS: We describe a prospective cohort study for comparing the immune and metabolic whole-blood markers in patients with sepsis, COVID-19 and other illnesses. Clinical phenotyping and blood culture test results will provide a reference standard to evaluate the performance of blood markers from the research sample analysis. Serial sampling of whole blood (50 µL each) will be collected from children admitted to intensive care and with an acute illness to follow time dependent changes in biomarkers. An integrated lipidomics and RNASeq transcriptomics analyses will be conducted to evaluate immune-metabolic networks that discriminate sepsis and COVID-19 from other acute illnesses. This study received approval for deferred consent. ETHICS AND DISSEMINATION: The study has received research ethics committee approval from the Yorkshire and Humber Leeds West Research Ethics Committee 2 (reference 20/YH/0214; IRAS reference 250612). Submission of study results for publication will involve making available all anonymised primary and processed data on public repository sites. TRIAL REGISTRATION NUMBER: NCT04904523.

Immune-metabolic adaptations in pregnancy: A potential stepping-stone to sepsis.

Physiological shifts during pregnancy predispose women to a higher risk of developing sepsis resulting from a maladapted host-response to infection. Insightful studies have delineated subtle point-changes to the immune system during pregnancy. Here, we present an overlay of these point-changes, asking what changes and when, at a physiological, cellular, and molecular systems-level in the context of sepsis. We identify distinct immune phases in pregnancy delineated by placental hormone-driven changes in homeostasis setpoints of the immune and metabolic systems that subtly mirrors changes observed in sepsis. We propose that pregnancy immune-metabolic setpoint changes impact feedback thresholds that increase risk for a maladapted host-response to infection and thus act as a stepping-stone to sepsis. Defining maternal immune-metabolic setpoint changes is not only vital for tailoring the right diagnostic tools for early management of maternal sepsis but will facilitate an unravelling of the pathophysiological pathways that predispose an individual to sepsis.

mSep: investigating physiological and immune-metabolic biomarkers in septic and healthy pregnant women to predict feto-maternal immune health - a prospective observational cohort study protocol.

INTRODUCTION: Maternal sepsis remains a leading cause of death in pregnancy. Physiological adaptations to pregnancy obscure early signs of sepsis and can result in delays in recognition and treatment. Identifying biomarkers that can reliably diagnose sepsis will reduce morbidity and mortality and antibiotic overuse. We have previously identified an immune-metabolic biomarker network comprising three pathways with a >99% accuracy for detecting bacterial neonatal sepsis. In this prospective study, we will describe physiological parameters and novel biomarkers in two cohorts-healthy pregnant women and pregnant women with suspected sepsis-with the aim of mapping pathophysiological drivers and evaluating predictive biomarkers for diagnosing maternal sepsis. METHODS AND ANALYSIS: Women aged over 18 with an ultrasound-confirmed pregnancy will be recruited to a pilot and two main study cohorts. The pilot will involve blood sample collection from 30 pregnant women undergoing an elective caesarean section. Cohort A will follow 100 healthy pregnant women throughout their pregnancy journey, with collection of blood samples from participants at routine time points in their pregnancy: week 12 'booking', week 28 and during labour. Cohort B will follow 100 pregnant women who present with suspected sepsis in pregnancy or labour and will have at least two blood samples taken during their care pathway. Study blood samples will be collected during routine clinical blood sampling. Detailed medical history and physiological parameters at the time of blood sampling will be recorded, along with the results of routine biochemical tests, including C reactive protein, lactate and white blood cell count. In addition, study blood samples will be processed and analysed for transcriptomic, lipidomic and metabolomic analyses and both qualitative and functional immunophenotyping. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the Wales Research Ethics Committee 2 (SPON1752-19, 30 October 2019). TRIAL REGISTRATION NUMBER: NCT05023954.

Challenging molecular dogmas in human sepsis using mathematical reasoning.

Sepsis is defined as a dysregulated host-response to infection, across all ages and pathogens. What defines a dysregulated state remains intensively researched but incompletely understood. Here, we dissect the meaning of this definition and its importance for the diagnosis and management of sepsis. We deliberate on pathophysiological features and dogmas that range from cytokine storms and immune paralysis to dormancy and altered homeostasis setpoints. Mathematical reasoning, used to test for plausibility, reveals three interlinked cardinal rules governing host-response trajectories in sepsis. Rule one highlights that the amplitude of the immune response while important is not sufficient and is strictly dependent on rule two, specifying bioenergetic capacity and are together dynamically driven by rule three, delineating stability and alterations in setpoints. We consider these rules and associated pathophysiological parameters for guiding data-science and artificial intelligence mining of multi-omics and big-data for improving the precision of diagnostic and therapeutic approaches to sepsis. FUNDING: PG funded by the European Regional Development Fund and Welsh Government (Ser Cymru programme - Project Sepsis).

Oxylipin metabolism is controlled by mitochondrial ß-oxidation during bacterial inflammation.

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial ß-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin ß-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by ß-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial ß-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

nSeP: immune and metabolic biomarkers for early detection of neonatal sepsis-protocol for a prospective multicohort study.

INTRODUCTION: Diagnosing neonatal sepsis is heavily dependent on clinical phenotyping as culture-positive body fluid has poor sensitivity, and existing blood biomarkers have poor specificity.A combination of machine learning, statistical and deep pathway biology analyses led to the identification of a tripartite panel of biologically connected immune and metabolic markers that showed greater than 99% accuracy for detecting bacterial infection with 100% sensitivity. The cohort study described here is designed as a large-scale clinical validation of this previous work. METHODS AND ANALYSIS: This multicentre observational study will prospectively recruit a total of 1445 newborn infants (all gestations)-1084 with suspected early-or late-onset sepsis, and 361 controls-over 4 years. A small volume of whole blood will be collected from infants with suspected sepsis at the time of presentation. This sample will be used for integrated transcriptomic, lipidomic and targeted proteomics profiling. In addition, a subset of samples will be subjected to cellular phenotype and proteomic analyses. A second sample from the same patient will be collected at 24 hours, with an opportunistic sampling for stool culture. For control infants, only one set of blood and stool sample will be collected to coincide with clinical blood sampling. Along with detailed clinical information, blood and stool samples will be analysed and the information will be used to identify and validate the efficacy of immune-metabolic networks in the diagnosis of bacterial neonatal sepsis and to identify new host biomarkers for viral sepsis. ETHICS AND DISSEMINATION: The study has received research ethics committee approval from the Wales Research Ethics Committee 2 (reference 19/WA/0008) and operational approval from Health and Care Research Wales. Submission of study results for publication will involve making available all anonymised primary and processed data on public repository sites. TRIAL REGISTRATION NUMBER: NCT03777670.

Sepsis target validation for repurposing and combining complement and immune checkpoint inhibition therapeutics.

Introduction: Sepsis is a disease that occurs due to an adverse immune response to infection by bacteria, viruses and fungi and is the leading pathway to death by infection. The hallmarks for maladapted immune reactions in severe sepsis, which contribute to multiple organ failure and death, are bookended by the exacerbated activation of the complement system to protracted T-cell dysfunction states orchestrated by immune checkpoint control. Despite major advances in our understanding of the condition, there remains to be either a definitive test or an effective therapeutic intervention.Areas covered: The authors consider a combinational drug therapy approach using new biologics, and mathematical modeling for predicting patient responses, in targeting innate and adaptive immune mediators underlying sepsis. Special consideration is given for emerging complement and immune checkpoint inhibitors that may be repurposed for sepsis treatment.Expert opinion: In order to overcome the challenges inherent to finding new therapies for the complex dysregulated host response to infection that drives sepsis, it is necessary to move away from monotherapy and promote precision for personalized combinatory therapies. Notably, combinatory therapy should be guided by predictive systems models of the immune-metabolic characteristics of an individual's disease progression.

LipidFinder 2.0: advanced informatics pipeline for lipidomics discovery applications.

SUMMARY: We present LipidFinder 2.0, incorporating four new modules that apply artefact filters, remove lipid and contaminant stacks, in-source fragments and salt clusters, and a new isotope deletion method which is significantly more sensitive than available open-access alternatives. We also incorporate a novel false discovery rate method, utilizing a target-decoy strategy, which allows users to assess data quality. A renewed lipid profiling method is introduced which searches three different databases from LIPID MAPS and returns bulk lipid structures only, and a lipid category scatter plot with color blind friendly pallet. An API interface with XCMS Online is made available on LipidFinder's online version. We show using real data that LipidFinder 2.0 provides a significant improvement over non-lipid metabolite filtering and lipid profiling, compared to available tools. AVAILABILITY AND IMPLEMENTATION: LipidFinder 2.0 is freely available at https://github.com/ODonnell-Lipidomics/LipidFinder and http://lipidmaps.org/resources/tools/lipidfinder. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mitoxantrone impairs proteasome activity and prompts early energetic and proteomic changes in HL-1 cardiomyocytes at clinically relevant concentrations.

Mitoxantrone (MTX) is used to treat several types of cancers and to improve neurological disability in multiple sclerosis. Unfortunately, cardiotoxicity is a severe and common adverse effect in MTX-treated patients. Herein, we aimed to study early and late mechanisms of MTX-induced cardiotoxicity using murine HL-1 cardiomyocytes. Cells were exposed to MTX (0.1, 1 or 10 µM) during short (2, 4, 6, or 12 h) or longer incubation periods (24 or 48 h). At earlier time points, (6 and 12 h) cytotoxicity was already observed for 1 and 10 µM MTX. Proteomic analysis of total protein extracts found 14 proteins with higher expression and 26 with lower expression in the cells exposed for 12 h to MTX (pH gradients 4-7 and 6-11). Of note, the expression of the regulatory protein 14-3-3 protein epsilon was increased by a factor of two and three, after exposure to 1 and 10 µM MTX, respectively. At earlier time-points, 10 µM MTX increased intracellular ATP levels, while decreasing media lactate levels. At later stages (24 and 48 h), MTX-induced cytotoxicity was concentration and time-dependent, according to the MTT reduction and lactate dehydrogenase leakage assays, while caspase-9, -8 and -3 activities increased at 24 h. Regarding cellular redox status, total glutathione increased in 1 µM MTX (24 h), and that increase was dependent on gamma-glutamylcysteine synthetase activity. Meanwhile, for both 1 and 10 µM MTX, oxidized glutathione was significantly higher than control at 48 h. Moreover, MTX was able to significantly decrease proteasomal chymotrypsin-like activity in a concentration and time-independent manner. In summary, MTX significantly altered proteomic, energetic and oxidative stress homeostasis in cardiomyocytes at clinically relevant concentrations and our data clearly demonstrate that MTX causes early cardiotoxicity that needs further study.

Innate immunology in COVID-19-a living review. Part I: viral entry, sensing and evasion.

The coronavirus infectious disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a world health concern and can cause severe disease and high mortality in susceptible groups. While vaccines offer a chance to treat disease, prophylactic and anti-viral treatments are still of vital importance, especially in context of the mutative ability of this group of viruses. Therefore, it is essential to elucidate the molecular mechanisms of viral entry, innate sensing and immune evasion of SARS-CoV-2, which control the triggers of the subsequent excessive inflammatory response. Viral evasion strategies directly target anti-viral immunity, counteracting host restriction factors and hijacking signalling pathways to interfere with interferon production. In Part I of this review, we examine SARS-CoV-2 viral entry and the described immune evasion mechanisms to provide a perspective on how the failure in initial viral sensing by infected cells can lead to immune dysregulation causing fatal COVID-19, discussed in Part II.

Innate immunology in COVID-19-a living review. Part II: dysregulated inflammation drives immunopathology.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a global health crisis and will likely continue to impact public health for years. As the effectiveness of the innate immune response is crucial to patient outcome, huge efforts have been made to understand how dysregulated immune responses may contribute to disease progression. Here we have reviewed current knowledge of cellular innate immune responses to SARS-CoV-2 infection, highlighting areas for further investigation and suggesting potential strategies for intervention. We conclude that in severe COVID-19 initial innate responses, primarily type I interferon, are suppressed or sabotaged which results in an early interleukin (IL)-6, IL-10 and IL-1ß-enhanced hyperinflammation. This inflammatory environment is driven by aberrant function of innate immune cells: monocytes, macrophages and natural killer cells dispersing viral pathogen-associated molecular patterns and damage-associated molecular patterns into tissues. This results in primarily neutrophil-driven pathology including fibrosis that causes acute respiratory distress syndrome. Activated leukocytes and neutrophil extracellular traps also promote immunothrombotic clots that embed into the lungs and kidneys of severe COVID-19 patients, are worsened by immobility in the intensive care unit and are perhaps responsible for the high mortality. Therefore, treatments that target inflammation and coagulation are promising strategies for reducing mortality in COVID-19.

Phospholipid membranes drive abdominal aortic aneurysm development through stimulating coagulation factor activity.

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA. Specifically, through activating coagulation, eoxPL either promoted or inhibited AAA depending on tissue localization. Ang II administration to ApoE-/- mice increased intravascular coagulation during AAA development. Lipidomics revealed large numbers of eoxPL formed within mouse and human AAA lesions. Deletion of eoxPL-generating enzymes (Alox12 or Alox15) or administration of the factor Xa inhibitor rivaroxaban significantly reduced AAA. Alox-deficient mice displayed constitutively dysregulated hemostasis, including a consumptive coagulopathy, characterized by compensatory increase in prothrombotic aminophospholipids (aPL) in circulating cell membranes. Intravenously administered procoagulant PL caused clotting factor activation and depletion, induced a bleeding defect, and significantly reduced AAA development. These data suggest that Alox deletion reduces AAA through diverting coagulation away from the vessel wall due to eoxPL deficiency, instead activating clotting factor consumption and depletion in the circulation. In mouse whole blood, ∼44 eoxPL molecular species formed within minutes of clot initiation. These were significantly elevated with ApoE-/- deletion, and many were absent in Alox-/- mice, identifying specific eoxPL that modulate AAA. Correlation networks demonstrated eoxPL belonged to subfamilies defined by oxylipin composition. Thus, procoagulant PL regulate AAA development through complex interactions with clotting factors. Modulation of the delicate balance between bleeding and thrombosis within either the vessel wall or circulation was revealed that can either drive or prevent disease development.