Hypoxia shapes the immune landscape in lung injury and promotes the persistence of inflammation.

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.

Cardiac Cell Therapy with Pluripotent Stem Cell-Derived Cardiomyocytes: What Has Been Done and What Remains to Do?

PURPOSE OF REVIEW: Exciting pre-clinical data presents pluripotent stem cell-derived cardiomyocytes (PSC-CM) as a novel therapeutic prospect following myocardial infarction, and worldwide clinical trials are imminent. However, despite notable advances, several challenges remain. Here, we review PSC-CM pre-clinical studies, identifying key translational hurdles. We further discuss cell production and characterization strategies, identifying markers that may help generate cells which overcome these barriers. RECENT FINDINGS: PSC-CMs can robustly repopulate infarcted myocardium with functional, force generating cardiomyocytes. However, current differentiation protocols produce immature and heterogenous cardiomyocytes, creating related issues such as arrhythmogenicity, immunogenicity and poor engraftment. Recent efforts have enhanced our understanding of cardiovascular developmental biology. This knowledge may help implement novel differentiation or gene editing strategies that could overcome these limitations. PSC-CMs are an exciting therapeutic prospect. Despite substantial recent advances, limitations of the technology remain. However, with our continued and increasing biological understanding, these issues are addressable, with several worldwide clinical trials anticipated in the coming years.

Neutrophil HIF-1α stabilization is augmented by mitochondrial ROS produced via the glycerol 3-phosphate shuttle.

Neutrophils are predominantly glycolytic cells that derive little ATP from oxidative phosphorylation; however, they possess an extensive mitochondrial network and maintain a mitochondrial membrane potential. Although studies have shown neutrophils need their mitochondria to undergo apoptosis and regulate NETosis, the metabolic role of the respiratory chain in these highly glycolytic cells is still unclear. Recent studies have expanded on the role of reactive oxygen species (ROS) released from the mitochondria as intracellular signaling molecules. Our study shows that neutrophils can use their mitochondria to generate ROS and that mitochondrial ROS release is increased in hypoxic conditions. This is needed for the stabilization of a high level of the critical hypoxic response factor and pro-survival protein HIF-1α in hypoxia. Further, we demonstrate that neutrophils use the glycerol 3-phosphate pathway as a way of directly regulating mitochondrial function through glycolysis, specifically to maintain polarized mitochondria and produce ROS. This illustrates an additional pathway by which neutrophils can regulate HIF-1α stability and will therefore be an important consideration when looking for treatments of inflammatory conditions in which HIF-1α activation and neutrophil persistence at the site of inflammation are linked to disease severity.

-------A type I IFN, prothrombotic hyperinflammatory neutrophil signature is distinct for COVID-19 ARDS--.

Background: Acute respiratory distress syndrome (ARDS) is a severe critical condition with a high mortality that is currently in focus given that it is associated with mortality caused by coronavirus disease 2019 (COVID-19). Neutrophils play a key role in the lung injury characteristic of non-COVID-19 ARDS and there is also accumulating evidence of neutrophil mediated lung injury in patients who succumb to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We undertook a functional proteomic and metabolomic survey of circulating neutrophil populations, comparing patients with COVID-19 ARDS and non-COVID-19 ARDS to understand the molecular basis of neutrophil dysregulation. Results: Expansion of the circulating neutrophil compartment and the presence of activated low and normal density mature and immature neutrophil populations occurs in ARDS, irrespective of cause. Release of neutrophil granule proteins, neutrophil activation of the clotting cascade and upregulation of the Mac-1 platelet binding complex with formation of neutrophil platelet aggregates is exaggerated in COVID-19 ARDS. Importantly, activation of components of the neutrophil type I interferon responses is seen in ARDS following infection with SARS-CoV-2, with associated rewiring of neutrophil metabolism, and the upregulation of antigen processing and presentation. Whilst dexamethasone treatment constricts the immature low density neutrophil population, it does not impact upon prothrombotic hyperinflammatory neutrophil signatures. Conclusions: Given the crucial role of neutrophils in ARDS and the evidence of a disordered myeloid response observed in COVID-19 patients, this work maps the molecular basis for neutrophil reprogramming in the distinct clinical entities of COVID-19 and non-COVID-19 ARDS.

Neutrophils Fuel Effective Immune Responses through Gluconeogenesis and Glycogenesis.

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.

Semaphorin 3F signaling actively retains neutrophils at sites of inflammation.

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.

Assessing the Efficacy of Dietary Selenomethionine Supplementation in the Setting of Cardiac Ischemia/Reperfusion Injury.

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury. This partly arises from the presence of activated phagocytes releasing myeloperoxidase (MPO) and its production of hypochlorous acid (HOCl). The dietary supplement selenomethionine (SeMet) has been shown to bolster endogenous antioxidant processes as well as readily react with MPO-derived oxidants. The aim of this study was to assess whether supplementation with SeMet could modulate the extent of cellular damage observed in an in vitro cardiac myocyte model exposed to (patho)-physiological levels of HOCl and an in vivo rat model of cardiac I/R injury. Exposure of the H9c2 cardiac myoblast cell line to HOCl resulted in a dose-dependent increase in necrotic cell death, which could be prevented by SeMet supplementation and was attributed to SeMet preventing the HOCl-induced loss of mitochondrial inner trans-membrane potential, and the associated cytosolic calcium accumulation. This protection was credited primarily to the direct oxidant scavenging ability of SeMet, with a minor contribution arising from the ability of SeMet to bolster cardiac myoblast glutathione peroxidase (GPx) activity. In vivo, a significant increase in selenium levels in the plasma and heart tissue were seen in male Wistar rats fed a diet supplemented with 2 mg kg-1 SeMet compared to controls. However, SeMet-supplementation demonstrated only limited improvement in heart function and did not result in better heart remodelling following I/R injury. These data indicate that SeMet supplementation is of potential benefit within pathological settings where excessive HOCl is known to be generated but has limited efficacy as a therapeutic agent for the treatment of heart attack.

Characterization of the cellular effects of myeloperoxidase-derived oxidants on H9c2 cardiac myoblasts.

Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury, which is in part due to the release of the enzyme myeloperoxidase (MPO) from activated infiltrating leukocytes, and the subsequent production of the oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Although exposure of various cell types to either oxidant is known to cause cellular dysfunction within a variety of pathological settings, the precise role of HOCl and HOSCN in the initiation of tissue damage evident following cardiac I/R injury remains unclear. In this study, we have employed the use of the cardiac myoblast cell line H9c2 as a model for cardiac myocytes and demonstrate that exposure to either oxidant elicits a dose-dependent increase in cytosolic calcium accumulation, depletion of the cellular thiol pool, reduction of glutathione (GSH) levels and loss of mitochondrial inner trans-membrane potential, concomitant with increased necrotic cell death. H9c2 cell recovery from the initial oxidant exposure involves the initiation of cell survival signalling pathways centred around Nrf2-antioxidant response element (ARE) and activator protein 1 (AP-1) activation, with cell survival accompanied by restoration of mitochondrial function following exposure to HOSCN, but not HOCl. These data highlight the cellular responses elicited by HOCl and HOSCN in cardiac myocytes furthering our understanding of the pathogenesis of oxidant injury following cardiac I/R injury.

Role of hypochlorous acid (HOCl) and other inflammatory mediators in the induction of macrophage extracellular trap formation.

The infiltration of activated leukocytes, including macrophages, at sites of inflammation and the formation and presence of hypochlorous acid (HOCl) are interlinked hallmarks of many debilitating disease processes, including atherosclerosis, arthritis, neurological and renal disease, diabetes and obesity. The production of extracellular traps by activated leukocytes in response to a range of inflammatory stimuli is increasingly recognised as an important process within a range of disease settings. We show that exposure of human monocyte-derived macrophages to pathophysiological levels of HOCl results in the dose-dependent extrusion of DNA and histones into the cellular supernatant, consistent with extracellular trap formation. Concurrent with, but independent of these findings, macrophage exposure to HOCl also resulted in an immediate and sustained cytosolic accumulation of Ca2+, culminating in the increased production of cytokines and chemokines. Polarisation of the macrophages prior to HOCl exposure revealed a greater propensity for inflammatory M1 macrophages to produce extracellular traps, whereas alternatively-activated M2 macrophages were less susceptible to HOCl insult. M1 macrophages also produced extracellular traps on exposure to phorbol myristate acetate (PMA), interleukin-8 (IL-8) and tumour necrosis factor α (TNFα). Taken together, these data indicate a potential role for macrophages in mediating extracellular trap formation, which may be relevant in pathological conditions characterised by chronic inflammation or excessive HOCl formation.