Search

Rationale: Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Objectives: Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. Methods: AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. Measurements and Main Results: We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration-derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. Conclusions: In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD. ispartof: AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE vol:207 issue:8 pages:998-1011 ispartof: location:United States status: published

Hypoxemia is a defining feature of acute respiratory distress syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, yet the resulting tissue hypoxia, and its impact on immune responses, is often neglected. In the present study, we have shown that ARDS patients were hypoxemic and monocytopenic within the first 48 h of ventilation. Monocytopenia was also observed in mouse models of hypoxic acute lung injury, in which hypoxemia drove the suppression of type I interferon signaling in the bone marrow. This impaired monopoiesis resulted in reduced accumulation of monocyte-derived macrophages and enhanced neutrophil-mediated inflammation in the lung. Administration of colony-stimulating factor 1 in mice with hypoxic lung injury rescued the monocytopenia, altered the phenotype of circulating monocytes, increased monocyte-derived macrophages in the lung and limited injury. Thus, tissue hypoxia altered the dynamics of the immune response to the detriment of the host and interventions to address the aberrant response offer new therapeutic strategies for ARDS.

Neutrophils are a critical element of host defence and are rapidly recruited to inflammatory sites. Such sites are frequently limited in oxygen and/or nutrient availability, presenting a metabolic challenge for infiltrating cells. Long believed to be uniquely dependent on glycolysis, it is now clear that neutrophils possess far greater metabolic plasticity than previously thought, with the capacity to generate energy stores and utilise extracellular proteins to fuel central carbon metabolism and biosynthetic activity. Out-with cellular energetics, metabolic programs have also been implicated in the production of neutrophils and their progenitors in the bone marrow compartment, activation of neutrophil anti-microbial responses, inflammatory and cell survival signalling cascades, and training of the innate immune response. Thus, understanding the mechanisms by which metabolic processes sustain changes in neutrophil effector functions and how these are subverted in disease states provides exciting new avenues for the treatment of dysfunctional neutrophilic inflammation which are lacking in clinical practice to date.

Acute Respiratory Distress Syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, has no cure. Hypoxemia is a defining feature, yet its impact on inflammation is often neglected. Patients with ARDS are monocytopenic early in the onset of the disease. Endotoxin or Streptococcus pneumoniae acute lung injury (ALI) in the context of hypoxia replicates this finding, through hypoxia-driven suppression of type I interferon signalling. This results in failed lung monocyte-derived interstitial macrophages (IM) niche expansion and unchecked neutrophilic inflammation. Administration of colony stimulating factor 1 (CSF1) rescues the monocytopenia, alters the circulating classical monocyte phenotype in hypoxic endotoxin-driven ALI and enables lung IM population expansion, thus limiting lung injury in endotoxin- and virally-induced hypoxic ALI. Hypoxia directly alters immune dynamics to the detriment of the host and manipulation of this aberrant response offers new therapeutic strategies for ARDS.

Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation

Limiting dysfunctional neutrophilic inflammation while preserving effective immunity requires a better understanding of the processes that dictate neutrophil function in the tissues. Quantitative mass-spectrometry identified how inflammatory murine neutrophils regulated expression of cell surface receptors, signal transduction networks, and metabolic machinery to shape neutrophil phenotypes in response to hypoxia. Through the tracing of labeled amino acids into metabolic enzymes, proinflammatory mediators, and granule proteins, we demonstrated that ongoing protein synthesis shapes the neutrophil proteome. To maintain energy supplies in the tissues, neutrophils consumed extracellular proteins to fuel central carbon metabolism. The physiological stresses of hypoxia and hypoglycemia, characteristic of inflamed tissues, promoted this extracellular protein scavenging with activation of the lysosomal compartment, further driving exploitation of the protein-rich inflammatory milieu. This study provides a comprehensive map of neutrophil proteomes, analysis of which has led to the identification of active catabolic and anabolic pathways that enable neutrophils to sustain synthetic and effector functions in the tissues. ispartof: JOURNAL OF CLINICAL INVESTIGATION vol:131 issue:10 ispartof: location:United States status: published

Summary Neutrophils can function and survive in injured and infected tissues, where oxygen and metabolic substrates are limited. Using radioactive flux assays and LC-MS tracing with U-13C glucose, glutamine, and pyruvate, we observe that neutrophils require the generation of intracellular glycogen stores by gluconeogenesis and glycogenesis for effective survival and bacterial killing. These metabolic adaptations are dynamic, with net increases in glycogen stores observed following LPS challenge or altitude-induced hypoxia. Neutrophils from patients with chronic obstructive pulmonary disease have reduced glycogen cycling, resulting in impaired function. Metabolic specialization of neutrophils may therefore underpin disease pathology and allow selective therapeutic targeting., Graphical Abstract, Highlights • Neutrophils utilize gluconeogenesis (GNG) to generate glycogen stores • Glycogenesis is essential for neutrophil survival and function • Defective glycogen cycling impairs neutrophil function in COPD • Glycogen cycling underpins the metabolic specialization of neutrophils, Neutrophils are required to meet their energy demands at inflamed sites where nutrients may be limited. Sadiku et al. provide evidence of a specialized metabolism that enables neutrophils to utilize glycogen cycling for energy production. This is essential for neutrophil function and survival, and dysregulation is associated with chronic disease states.

Severe Covid-19 is associated with elevated plasma Factor V (FV) and increased risk of thromboembolism. We report that neutrophils, T regulatory cells (Tregs), and monocytes from patients with severe Covid-19 express FV, and expression correlates with T cell lymphopenia. In vitro full length FV, but not FV activated by thrombin cleavage, suppresses T cell proliferation. Increased and prolonged FV expression by cells of the innate and adaptive immune systems may contribute to lymphopenia in severe Covid-19. Activation by thrombin destroys the immunosuppressive properties of FV. Anticoagulation in Covid-19 patients may have the unintended consequence of suppressing the adaptive immune system.

SummaryUnderstanding the mechanisms by which infection with SARS-CoV-2 leads to acute respiratory distress syndrome (ARDS) is of significant clinical interest given the mortality associated with severe and critical coronavirus induced disease 2019 (COVID-19). Neutrophils play a key role in the lung injury characteristic of non-COVID-19 ARDS, but a relative paucity of these cells is observed at post-mortem in lung tissue of patients who succumb to infection with SARS-CoV-2. With emerging evidence of a dysregulated innate immune response in COVID-19, we undertook a functional proteomic survey of circulating neutrophil populations, comparing patients with COVID-19 ARDS, non-COVID-19 ARDS, moderate COVID-19, and healthy controls. We observe that expansion of the circulating neutrophil compartment and the presence of activated low and normal density mature and immature neutrophil populations occurs in both COVID-19 and non-COVID-19 ARDS. In contrast, release of neutrophil granule proteins, neutrophil activation of the clotting cascade and formation of neutrophil platelet aggregates is significantly increased in COVID-19 ARDS. Importantly, activation of components of the neutrophil type I IFN responses is specific to infection with SARS-CoV-2 and linked to metabolic rewiring. Together this work highlights how differential activation of circulating neutrophil populations may contribute to the pathogenesis of ARDS, identifying processes that are specific to COVID-19 ARDS.

Neutrophilic inflammation is central to disease pathogenesis, for example, in chronic obstructive pulmonary disease, yet the mechanisms that retain neutrophils within tissues remain poorly understood. With emerging evidence that axon guidance factors can regulate myeloid recruitment and that neutrophils can regulate expression of a class 3 semaphorin, SEMA3F, we investigated the role of SEMA3F in inflammatory cell retention within inflamed tissues. We observed that neutrophils upregulate SEMA3F in response to proinflammatory mediators and following neutrophil recruitment to the inflamed lung. In both zebrafish tail injury and murine acute lung injury models of neutrophilic inflammation, overexpression of SEMA3F delayed inflammation resolution with slower neutrophil migratory speeds and retention of neutrophils within the tissues. Conversely, constitutive loss of sema3f accelerated egress of neutrophils from the tail injury site in fish, whereas neutrophil-specific deletion of Sema3f in mice resulted in more rapid neutrophil transit through the airways, and significantly reduced time to resolution of the neutrophilic response. Study of filamentous-actin (F-actin) subsequently showed that SEMA3F-mediated retention is associated with F-actin disassembly. In conclusion, SEMA3F signaling actively regulates neutrophil retention within the injured tissues with consequences for neutrophil clearance and inflammation resolution.

Introduction and objectives Acute Respiratory Distress Syndrome (ARDS) is characterised by neutrophil driven alveolar and vascular injury. Alveolar damage is associated with worsening hypoxia and increased mortality. Specific therapies targeting neutrophilic inflammation are lacking, in part due to the challenge of limiting pathological inflammation while preserving immunity. We sought to characterise the effect of hypoxia on neutrophil driven ARDS and to define the mechanisms underlying the hypoxic phenotype. Methods We used a mouse model of lipopolysaccharide (LPS)-induced ARDS with mice subsequently housed in either room air or in a hypoxic chamber (FiO2 10%). High resolution mass spectrometry was used to define the proteome of normoxic and hypoxic inflammatory lung neutrophils. Results Exposure to hypoxia in the ARDS model resulted in increased morbidity with significant hypothermia and increased lung injury. Lung damage was associated with enhanced neutrophil degranulation, with elevated levels of elastase and MMP9 in the bronchoalveolar lavage (BAL) of hypoxic mice. Tissue injury was independent of neutrophil number suggesting that hypoxia results in a fundamental change in neutrophil phenotype and, indeed, a distinct and hyperinflammatory hypoxic proteomic signature was observed. More specifically, upregulation of inflammatory receptors including GM-CSF, TNF-alpha and formylated peptide receptors were identified as drivers of enhanced in-vivo neutrophil degranulation in hypoxia. Analysis of the proteome of these inflammatory tissue neutrophils also provided insights into the processes and pathways which are active, highlighting the critical role of metabolic pathways in neutrophil function. Hypoxia was shown to drive metabolic adaptations with enhanced biosynthesis of inflammatory mediators further contributing to the hyperinflammatory phenotype. Upregulation of the lysosome and suppression of the nutrient sensing complex mTORC1 were shown to regulate these pathways in hypoxic neutrophils. Conclusions Neutrophilic inflammation and hypoxia frequently co-exist, and we have demonstrated that hypoxia results in a damaging, hyperinflammatory phenotype in tissue neutrophils. Proteomic analysis identifies upregulation of inflammatory receptors and metabolic adaptations in hypoxic neutrophils as key drivers of this phenotype. Characterisation of these pathways driving harmful inflammation in the hypoxic lung identify new potential therapeutic targets in ARDS.

Effective host responses to injury and infection require both rapid recruitment of neutrophils into tissues and timely inflammation resolution. Research currently focuses on initiation and resolution as distinct phases of inflammation and neutrophil survival as a key determinant of the inflammatory response. Less focus has been placed on mechanisms retaining viable neutrophils at inflamed sites and thus contributing to ongoing inflammation. This may be of therapeutic importance given the numerical dominance of viable over apoptotic neutrophils in inflamed tissue, even during inflammation resolution. Semaphorins are proteins that regulate motility in a variety of cell types and systems. We observed that neutrophils regulate Sema3F transcript abundance, which is expressed in the airway myeloid cell population of COPD patients. We then questioned whether Sema3F could regulate neutrophil migration and define the magnitude of the inflammatory response. Not only do inflammatory neutrophils express the class 3 Semaphorin, Sema3F and its obligatory co-receptor Neuropilin 2 (NRP2) but expression of both is further induced by bacterial Lipopolysaccharide (LPS) and other inflammatory stimuli. In a murine model of LPS-induced acute lung injury, we describe a phenotype where neutrophil specific knockdown of Sema3F both increases neutrophil recruitment to the airways 6 hours following LPS challenge (***p=0.0079, n=9) and enhances the rate of inflammation resolution (wild-type airway neutrophil count T50=42 hours, Sema3F knockout T50=32.9 hours). Conversely, intra-tracheal instillation of Sema3F in wild-type mice retains neutrophils at the injury site following maximal recruitment (p=0.046, n=8). In vitro experiments using human neutrophils demonstrate Sema3F reduces neutrophil chemotaxis producing ‘rounder cells’, phagocytosis is unaffected but interestingly respiratory burst is enhanced (*p=0.0117, n=3). Using confocal microscopy and image analysis software we identified the location of the neutrophils in the murine lung interstitium following LPS induced lung injury. We show that intra-tracheal Sema3F holds the recruited neutrophils in the alveolar space (*p

Cells sense and respond to hypoxia through the activity of the transcription factor HIF (hypoxia-inducible factor) and its regulatory hydroxylases, the prolyl hydroxylase domain enzymes (PHDs). Multiple isoforms of HIFs and PHDs exist, and isoform-selective roles have been identified in the context of the inflammatory environment, which is itself frequently hypoxic. Recent advances in the field have highlighted the complexity of this system, particularly with regards to the cell and context-specific activity of HIFs and PHDs. Because novel therapeutic agents which regulate this pathway are nearing the clinic, understanding the role of HIFs and PHDs in inflammation outcomes is an essential step in avoiding off-target effects and, crucially, in developing new anti-inflammatory strategies.

Tissue damage and infection are deemed likewise triggers of innate immune responses. But whereas neutrophil responses to microbes are generally protective, neutrophil recruitment into damaged tissues without infection is deleterious. Why neutrophils respond to tissue damage and not just to microbes is unknown. Is it a flaw of the innate immune system that persists because evolution did not select against it, or does it provide a selective advantage? Here, we dissect the contribution of tissue damage signaling to antimicrobial immune responses in a live vertebrate. By intravital imaging of zebrafish larvae, a powerful model for innate immunity, we show that prevention of tissue damage signaling upon microbial ear infection abrogates leukocyte chemotaxis, and reduces animal survival, at least in part, through suppression of cytosolic phospholipase A(2) (cPla(2)), which integrates tissue damage- and microbe-derived cues. Thus, microbial cues are insufficient, and damage signaling is essential for antimicrobial neutrophil responses in zebrafish.

Fully activated innate immune cells are required for effective responses to infection, but their prompt deactivation and removal are essential for limiting tissue damage. Here, we have identified a critical role for the prolyl hydroxylase enzyme Phd2 in maintaining the balance between appropriate, predominantly neutrophil-mediated pathogen clearance and resolution of the innate immune response. We demonstrate that myeloid-specific loss of Phd2 resulted in an exaggerated inflammatory response to Streptococcus pneumonia, with increases in neutrophil motility, functional capacity, and survival. These enhanced neutrophil responses were dependent upon increases in glycolytic flux and glycogen stores. Systemic administration of a HIF-prolyl hydroxylase inhibitor replicated the Phd2-deficient phenotype of delayed inflammation resolution. Together, these data identify Phd2 as the dominant HIF-hydroxylase in neutrophils under normoxic conditions and link intrinsic regulation of glycolysis and glycogen stores to the resolution of neutrophil-mediated inflammatory responses. These results demonstrate the therapeutic potential of targeting metabolic pathways in the treatment of inflammatory disease. ispartof: Journal of Clinical Investigation vol:127 issue:9 pages:3407-3420 ispartof: location:United States status: published

Hypoxia and bacterial infection frequently coexist, in both acute and chronic clinical settings, and typically result in adverse clinical outcomes. To ameliorate this morbidity, we investigated the interaction between hypoxia and the host response. In the context of acute hypoxia, both Staphylococcus aureus and Streptococcus pneumoniae infections rapidly induced progressive neutrophil-mediated morbidity and mortality, with associated hypothermia and cardiovascular compromise. Preconditioning animals through longer exposures to hypoxia, before infection, prevented these pathophysiological responses and profoundly dampened the transcriptome of circulating leukocytes. Specifically, perturbation of hypoxia-inducible factor (HIF) pathway and glycolysis genes by hypoxic preconditioning was associated with reduced leukocyte glucose utilization, resulting in systemic rescue from a global negative energy state and myocardial protection. Thus, we demonstrate that hypoxia preconditions the innate immune response and determines survival outcomes after bacterial infection through suppression of HIF-1α and neutrophil metabolism. In the context of systemic or tissue hypoxia, therapies that target the host response could improve infection-associated morbidity and mortality.

To develop and test a set of measures of quality of care in the process of sepsis management, to determine the inter-rater reliability of case-note review in assessment of these measures and to assess our current standard of care.Five measures of process of care and one of outcome were identified from the literature review and previous experience. Failure modes and effects analysis was used by a multidisciplinary team to validate these measures and prioritize them in terms of associated risk. Forty sets of case notes were reviewed by two independent teams and the inter-rater reliability was determined using observed percentage agreement and the kappa statistic. We used the data to calculate the proportion of patients in whom we are currently meeting targets for good quality of care.The multidisciplinary team did not identify any additional areas of concern and assigned the highest risk priority to a delay of over 4 h from recognition of sepsis to antibiotic administration. The inter-rater agreement was80% for four of the measures, but was only 62.5% for appropriateness of antibiotic therapy. Room for improvement in practice exists, for example, antibiotic administration within 4 h was not achieved in 40% of patients.Four of our five measures of care are suitable for use in assessing the effect of interventions aimed at improving sepsis management, with at least moderate inter-rater reliability. Specific areas where increased clarity should improve agreement further have been identified.