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.

ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system.

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.

Getting DAMP(s) Wets the Whistle for Neutrophil Recruitment.

Neutrophil recruitment in response to pathogen invasion is mediated through "self" tissue damage signals (DAMPs) and pathogen associated signals (PAMPs). In this issue of Immunity, Huang and Niethammer, (2018) demonstrate that DAMP signaling is a prerequisite for neutrophil recruitment.

The emerging role for metabolism in fueling neutrophilic inflammation.

Neutrophils are a critical element of host defense 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 utilize 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 antimicrobial responses, inflammatory and cell survival signaling 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.

NRF2 Activation Reprograms Defects in Oxidative Metabolism to Restore Macrophage Function in Chronic Obstructive Pulmonary Disease.

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.

IL4Rα Signaling Abrogates Hypoxic Neutrophil Survival and Limits Acute Lung Injury Responses In Vivo.

Rationale: Acute respiratory distress syndrome is defined by the presence of systemic hypoxia and consequent on disordered neutrophilic inflammation. Local mechanisms limiting the duration and magnitude of this neutrophilic response remain poorly understood. Objectives: To test the hypothesis that during acute lung inflammation tissue production of proresolution type 2 cytokines (IL-4 and IL-13) dampens the proinflammatory effects of hypoxia through suppression of HIF-1α (hypoxia-inducible factor-1α)-mediated neutrophil adaptation, resulting in resolution of lung injury. Methods: Neutrophil activation of IL4Ra (IL-4 receptor α) signaling pathways was explored ex vivo in human acute respiratory distress syndrome patient samples, in vitro after the culture of human peripheral blood neutrophils with recombinant IL-4 under conditions of hypoxia, and in vivo through the study of IL4Ra-deficient neutrophils in competitive chimera models and wild-type mice treated with IL-4. Measurements and Main Results: IL-4 was elevated in human BAL from patients with acute respiratory distress syndrome, and its receptor was identified on patient blood neutrophils. Treatment of human neutrophils with IL-4 suppressed HIF-1α-dependent hypoxic survival and limited proinflammatory transcriptional responses. Increased neutrophil apoptosis in hypoxia, also observed with IL-13, required active STAT signaling, and was dependent on expression of the oxygen-sensing prolyl hydroxylase PHD2. In vivo, IL-4Ra-deficient neutrophils had a survival advantage within a hypoxic inflamed niche; in contrast, inflamed lung treatment with IL-4 accelerated resolution through increased neutrophil apoptosis. Conclusions: We describe an important interaction whereby IL4Rα-dependent type 2 cytokine signaling can directly inhibit hypoxic neutrophil survival in tissues and promote resolution of neutrophil-mediated acute lung injury.

Promotion and views on tinnitus self-help within United Kingdom national health service audiology departments.

Self-help has the potential to provide low-cost and effective ways of improving access to psychological support for people with tinnitus. When developing and evaluating resources it is important to consider issues that may influence successful implementation. This Survey explored clinicians' use and views on self-help, and barriers to implementation. An online survey was distributed to 220 UK audiology departments. One-hundred and twenty-four clinicians responded to the survey (91 audiologists, 32 hearing therapists, 1 clinical psychologist), each representing a different tinnitus department. Two-thirds of respondents reported providing or recommending self-help resources. Potential benefits were patient empowerment and providing a means of engaging patients in their care. Almost half of respondents felt that there is insufficient training or guidance for clinicians on using or promoting self-help. Clinicians felt that for patients with low-level tinnitus severity, self-help may reduce the number of audiology appointments required. For patients with more complex needs self-help may be useful to engage with between clinical appointments. Further research is needed to determine effectiveness, who benefits, and by what mechanism, before clinicians can confidently recommend or implement self-help. Clinicians will benefit from formal guidance on promoting and supporting use of self-help for tinnitus.

Effects of seasonal deposition-erosion cycle on sedimentary organic carbon remineralization and oxygen consumption in a large-river delta-front estuary.

Seasonal sediment deposition-erosion events are dominant drivers of particle-solute dynamics in large-river delta-front estuaries (LDEs), but their influence on elemental cycles is not yet fully understood. To better constrain the role of deposition-erosion events on elemental cycling in LDEs, benthic fluxes of dissolved inorganic carbon (DIC), oxygen, and pore-water solute profiles were measured over different seasons in the Changjiang LDE. Benthic DIC efflux (23.4 ± 6.0 mmol C m-2 d-1) was greater than oxygen influx (7.5 ± 2.0 mmol O2 m-2 d-1) in summer but less in winter (7.7 ± 1.2 mmol C m-2 d-1 and 10.1 ± 1.5 mmol O2 m-2 d-1, respectively). The additional oxygen consumption in sediments in winter was likely due to the oxidation of inorganic diagenetic reductive products (IDRP) (e.g., NH4+, Fe2+, and Mn2+) in deeper sediments exposed by erosion, which resulted in the development of an "oxygen debt". Sedimentary oxygen respiration accounted for at least 48 % of total oxygen consumption (oxygen consumption in both water column and sediment) in winter and was significantly greater than in summer (∼15 %); this highlighted the importance of winter sediment erosion in oxygen depletion. In addition to IDRP oxidation, the remineralization of resuspended sedimentary organic carbon in water column also contributed to the oxygen consumption. The global dataset on benthic DIC and oxygen fluxes provides evidence that the "oxygen debt" is likely to be widespread in LDEs, exerting a significant impact on global carbon and oxygen cycling.

IS-PRM-based peptide targeting informed by long-read sequencing for alternative proteome detection.

Alternative splicing is a major contributor of transcriptomic complexity, but the extent to which transcript isoforms are translated into stable, functional protein isoforms is unclear. Furthermore, detection of relatively scarce isoform-specific peptides is challenging, with many protein isoforms remaining uncharted due to technical limitations. Recently, a family of advanced targeted MS strategies, termed internal standard parallel reaction monitoring (IS-PRM), have demonstrated multiplexed, sensitive detection of pre-defined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here, we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (LR RNAseq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides, which are specific to individual gene product isoforms, serve as "triggers" and "targets" in the IS-PRM method, Tomahto. Using the model human stem cell line WTC11, LR RNAseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic "trigger" peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest, predicted to contain corresponding endogenous "target" peptides. Compared to DDA mode, Tomahto increased detectability of isoforms by 3.6-fold, resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This LR RNA seq-informed Tomahto targeted approach, called LRP-IS-PRM, is a new modality for generating protein-level evidence of alternative isoforms - a critical first step in designing functional studies and eventually clinical assays.

IS-PRM-Based Peptide Targeting Informed by Long-Read Sequencing for Alternative Proteome Detection.

Alternative splicing is a major contributor of transcriptomic complexity, but the extent to which transcript isoforms are translated into stable, functional protein isoforms is unclear. Furthermore, detection of relatively scarce isoform-specific peptides is challenging, with many protein isoforms remaining uncharted due to technical limitations. Recently, a family of advanced targeted MS strategies, termed internal standard parallel reaction monitoring (IS-PRM), have demonstrated multiplexed, sensitive detection of predefined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here, we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (lrRNA-seq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides, which are specific to individual gene product isoforms, serve as "triggers" and "targets" in the IS-PRM method, Tomahto. Using the model human stem cell line WTC11, LR RNaseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic "trigger" peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest, predicted to contain corresponding endogenous "target" peptides. Compared to DDA mode, Tomahto increased detectability of isoforms by 3.6-fold, resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This lrRNA-seq-informed Tomahto targeted approach is a new modality for generating protein-level evidence of alternative isoforms─a critical first step in designing functional studies and eventually clinical assays.

Control of mitophagy initiation and progression by the TBK1 adaptors NAP1 and SINTBAD.

Mitophagy preserves overall mitochondrial fitness by selectively targeting damaged mitochondria for degradation. The regulatory mechanisms that prevent PTEN-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase Parkin (PINK1/Parkin)-dependent mitophagy and other selective autophagy pathways from overreacting while ensuring swift progression once initiated are largely elusive. Here, we demonstrate how the TBK1 (TANK-binding kinase 1) adaptors NAP1 (NAK-associated protein 1) and SINTBAD (similar to NAP1 TBK1 adaptor) restrict the initiation of OPTN (optineurin)-driven mitophagy by competing with OPTN for TBK1. Conversely, they promote the progression of nuclear dot protein 52 (NDP52)-driven mitophagy by recruiting TBK1 to NDP52 and stabilizing its interaction with FIP200. Notably, OPTN emerges as the primary recruiter of TBK1 during mitophagy initiation, which in return boosts NDP52-mediated mitophagy. Our results thus define NAP1 and SINTBAD as cargo receptor rheostats, elevating the threshold for mitophagy initiation by OPTN while promoting the progression of the pathway once set in motion by supporting NDP52. These findings shed light on the cellular strategy to prevent pathway hyperactivity while still ensuring efficient progression.

Safety of bendamustine for the treatment of indolent non-Hodgkin lymphoma: a UK real-world experience.

ABSTRACT: Bendamustine is among the most effective chemotherapeutics for indolent B-cell non-Hodgkin lymphomas (iNHL), but trial reports of significant toxicity, including opportunistic infections and excess deaths, led to prescriber warnings. We conducted a multicenter observational study evaluating bendamustine toxicity in real-world practice. Patients receiving at least 1 dose of bendamustine with/without rituximab (R) for iNHL were included. Demographics, lymphoma and treatment details, and grade 3 to 5 adverse events (AEs) were analyzed and correlated. In total, 323 patients were enrolled from 9 National Health Service hospitals. Most patients (96%) received bendamustine-R, and 46%, R maintenance. Overall, 21.7% experienced serious AEs (SAE) related to treatment, including infections in 12%, with absolute risk highest during induction (63%), maintenance (20%), and follow-up (17%) and the relative risk highest during maintenance (54%), induction (34%), and follow-up (28%). Toxicity led to permanent treatment discontinuation for 13% of patients, and 2.8% died of bendamustine-related infections (n = 5), myelodysplastic syndrome (n = 3), and cardiac disease (n = 1). More SAEs per patient were reported in patients with mantle cell lymphoma, poor preinduction performance status (PS), poor premaintenance PS, and abnormal preinduction total globulins and in those receiving growth factors. Use of antimicrobial prophylaxis was variable, and 3 of 10 opportunistic infections occurred despite prophylaxis. In this real-world analysis, bendamustine-related deaths and treatment discontinuation were similar to those of trial populations of younger, fitter patients. Poor PS, mantle cell histology, and maintenance R were potential risk factors. Infections, including late onset events, were the most common treatment-related SAE and cause of death, warranting extended antimicrobial prophylaxis and infectious surveillance, especially for maintenance-treated patients.

Impacts of diethylhexyl phthalate and overfeeding on physical fitness and lipid mobilization in Danio rerio (zebrafish).

As the prevalence of obesity has steadily increased on a global scale, research has shifted to explore potential contributors to this pandemic beyond overeating and lack of exercise. Environmental chemical contaminants, known as obesogens, alter metabolic processes and exacerbate the obese phenotype. Diethylhexyl phthalate (DEHP) is a common chemical plasticizer found in medical supplies, food packaging, and polyvinyl materials, and has been identified as a probable obesogen. This study investigated the hypothesis that co-exposure to DEHP and overfeeding would result in decreased lipid mobilization and physical fitness in Danio rerio (zebrafish). Four treatment groups were randomly assigned: Regular Fed (control, 10 mg/fish/day with 0 mg/kg DEHP), Overfed (20 mg/fish/day with 0 mg/kg DEHP), Regular Fed + DEHP (10 mg/fish/day with 3 mg/kg DEHP), Overfed + DEHP (20 mg/fish/day with 3 mg/kg DEHP). After 24 weeks, swim tunnel assays were conducted on half of the zebrafish from each treatment to measure critical swimming speeds (Ucrit); the other fish were euthanized without swimming. Body mass index (BMI) was measured, and tissues were collected for blood lipid characterization and gene expression analyses. Co-exposure to DEHP and overfeeding decreased swim performance as measured by Ucrit. While no differences in blood lipids were observed with DEHP exposure, differential expression of genes related to lipid metabolism and utilization in the gastrointestinal and liver tissue suggests alterations in metabolism and lipid packaging, which may impact utilization and ability to mobilize lipid reserves during physical activity following chronic exposures.

Coagulation factor V is a T-cell inhibitor expressed by leukocytes in COVID-19.

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 in vitro. Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system.

Serum response factor promotes resilience to chronic social stress through the induction of DeltaFosB.

The molecular mechanisms underlying stress- and drug-induced neuronal adaptations are incompletely understood. One molecule implicated in such adaptations is ΔFosB, a transcription factor that accumulates in the rodent nucleus accumbens (NAc), a key brain reward region, in response to either chronic stress or repeated exposure to drugs of abuse. The upstream transcriptional mechanisms controlling ΔFosB induction by these environmental stimuli remain elusive. Here, we identify the activity-dependent transcription factor, serum response factor (SRF), as a novel upstream mediator of stress-, but not cocaine-, induced ΔFosB. SRF is downregulated in NAc of both depressed human patients and in mice chronically exposed to social defeat stress. This downregulation of SRF is absent in resilient animals. Through the use of inducible mutagenesis, we show that stress-mediated induction of ΔFosB, which occurs predominantly in resilient mice, is dependent on SRF expression in this brain region. Furthermore, NAc-specific genetic deletion of SRF promotes a variety of prodepressant- and proanxiety-like phenotypes and renders animals more sensitive to the deleterious effects of chronic stress. In contrast, we demonstrate that SRF does not play a role in ΔFosB accumulation in NAc in response to chronic cocaine exposure. Furthermore, NAc-specific knock-out of SRF has no effect on cocaine-induced behaviors, indicating that chronic social defeat stress and repeated cocaine exposure regulate ΔFosB accumulation and behavioral sensitivity through independent mechanisms.

Hypoxia drives murine neutrophil protein scavenging to maintain central carbon metabolism.

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.

-------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.