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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.

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

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.