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Altered redox biology challenges all cells, with compensatory responses often determining a cell's fate. When 15 lipoxygenase 1 (15LO1), a lipid-peroxidizing enzyme abundant in asthmatic human airway epithelial cells (HAECs), binds phosphatidylethanolamine-binding protein 1 (PEBP1), hydroperoxy-phospholipids, which drive ferroptotic cell death, are generated. Peroxidases, including glutathione peroxidase 4 (GPX4), metabolize hydroperoxy-phospholipids to hydroxy derivatives to prevent ferroptotic death, but consume reduced glutathione (GSH). The cystine transporter SLC7A11 critically restores/maintains intracellular GSH. We hypothesized that high 15LO1, PEBP1, and GPX4 activity drives abnormal asthmatic redox biology, evidenced by lower bronchoalveolar lavage (BAL) fluid and intraepithelial cell GSH:oxidized GSH (GSSG) ratios, to enhance type 2 (T2) inflammatory responses. GSH, GSSG (enzymatic assays), 15LO1, GPX4, SLC7A11, and T2 biomarkers (Western blot and RNA-Seq) were measured in asthmatic and healthy control (HC) cells and fluids, with siRNA knockdown as appropriate. GSSG was higher and GSH:GSSG lower in asthmatic compared with HC BAL fluid, while intracellular GSH was lower in asthma. In vitro, a T2 cytokine (IL-13) induced 15LO1 generation of hydroperoxy-phospholipids, which lowered intracellular GSH and increased extracellular GSSG. Lowering GSH further by inhibiting SLC7A11 enhanced T2 inflammatory protein expression and ferroptosis. Ex vivo, redox imbalances corresponded to 15LO1 and SLC7A11 expression, T2 biomarkers, and worsened clinical outcomes. Thus, 15LO1 pathway-induced redox biology perturbations worsen T2 inflammation and asthma control, supporting 15LO1 as a therapeutic target.

Antigen-presenting cells (APCs) occupy diverse anatomical tissues, but their tissue-restricted homeostasis remains poorly understood. Here, working with mouse models of inflammation, we found that mechanistic target of rapamycin (mTOR)–dependent metabolic adaptation was required at discrete locations. mTOR was dispensable for dendritic cell (DC) homeostasis in secondary lymphoid tissues but necessary to regulate cellular metabolism and accumulation of CD103⁺ DCs and alveolar macrophages in lung. Moreover, while numbers of mTOR-deficient lung CD11b⁺ DCs were not changed, they were metabolically reprogrammed to skew allergic inflammation from eosinophilic T helper cell 2 (TH2) to neutrophilic TH17 polarity. The mechanism for this change was independent of translational control but dependent on inflammatory DCs, which produced interleukin-23 and increased fatty acid oxidation. mTOR therefore mediates metabolic adaptation of APCs in distinct tissues, influencing the immunological character of allergic inflammation.

Bacterial pneumonia is a significant healthcare burden worldwide. Failure to resolve inflammation after infection precipitates lung injury and an increase in morbidity and mortality. Gram-negative bacteria are common in pneumonia and increased levels of the mito-damage-associated molecular pattern (DAMP) cardiolipin can be detected in the lungs. Here we show that mice infected with Klebsiella pneumoniae develop lung injury with accumulation of cardiolipin. Cardiolipin inhibits resolution of inflammation by suppressing production of anti-inflammatory IL-10 by lung CD11b + Ly6G int Ly6C lo F4/80 + cells. Cardiolipin induces PPARγ SUMOylation, which causes recruitment of a repressive NCOR/HDAC3 complex to the IL-10 promoter, but not the TNF promoter, thereby tipping the balance towards inflammation rather than resolution. Inhibition of HDAC activity by sodium butyrate enhances recruitment of acetylated histone 3 to the IL-10 promoter and increases the concentration of IL-10 in the lungs. These findings identify a mechanism of persistent inflammation during pneumonia and indicate the potential of HDAC inhibition as a therapy. Non-resolving bacterial pneumonia results in lung tissue damage owing to overactive inflammation. Here the authors show that the mitochondrial DAMP cardiolipin contributes to persistent inflammation by SUMOylating PPARγ, which promotes binding of the corepressor NCOR/HDAC3 complex to the IL-10 promoter.

Respiratory infection by Pseudomonas aeruginosa , common in hospitalized immunocompromised and immunocompetent ventilated patients, can be life-threatening because of antibiotic resistance. This raises the question of whether the host’s immune system can be educated to combat this bacterium. Here we show that prior exposure to a single low dose of lipopolysaccharide (LPS) protects mice from a lethal infection by P. aeruginosa . LPS exposure trained the innate immune system by promoting expansion of neutrophil and interstitial macrophage populations distinguishable from other immune cells with enrichment of gene sets for phagocytosis- and cell-killing-associated genes. The cell-killing gene set in the neutrophil population uniquely expressed Lgals3 , which encodes the multifunctional antibacterial protein, galectin-3. Intravital imaging for bacterial phagocytosis, assessment of bacterial killing and neutrophil-associated galectin-3 protein levels together with use of galectin-3-deficient mice collectively highlight neutrophils and galectin-3 as central players in LPS-mediated protection. Patients with acute respiratory failure revealed significantly higher galectin-3 levels in endotracheal aspirates (ETAs) of survivors compared to non-survivors, galectin-3 levels strongly correlating with a neutrophil signature in the ETAs and a prognostically favorable hypoinflammatory plasma biomarker subphenotype. Taken together, our study provides impetus for harnessing the potential of galectin-3-expressing neutrophils to protect from lethal infections and respiratory failure. This study reports training by lipopolysaccharide to expand neutrophils expressing the anti-bacterial galectin-3 protein defending mice from a lethal bacterial infection, a similar signature associated with survivors of respiratory failure in humans.

A stable mitochondrial pool is crucial for healthy cell function and survival. Altered redox biology can adversely affect mitochondria through induction of a variety of cell death and survival pathways, yet the understanding of mitochondria and their dysfunction in primary human cells and in specific disease states, including asthma, is modest. Ferroptosis is traditionally considered an iron dependent, hydroperoxy-phospholipid executed process, which induces cytosolic and mitochondrial damage to drive programmed cell death. However, in this report we identify a lipoxygenase orchestrated, compartmentally-targeted ferroptosis-associated peroxidation process which occurs in a subpopulation of dysfunctional mitochondria, without promoting cell death. Rather, this mitochondrial peroxidation process tightly couples with PTEN-induced kinase (PINK)−1(PINK1)-Parkin-Optineurin mediated mitophagy in an effort to preserve the pool of functional mitochondria and prevent cell death. These combined peroxidation processes lead to altered epithelial cell phenotypes and loss of ciliated cells which associate with worsened asthma severity. Ferroptosis-targeted interventions of this process could preserve healthy mitochondria, reverse cell phenotypic changes and improve disease outcomes. Altered redox biology causes mitochondrial dysfunction, yet mechanisms and implications vary. Here, the authors identify 15 lipoxygenase-linked mitochondrial ferroptosis and mitophagy driving epithelial cell differentiation and asthma outcomes.

The severity of bacterial pneumonia can be worsened by impaired innate immunity resulting in ineffective pathogen clearance. We describe a mitochondrial protein, aspartyl-tRNA synthetase (DARS2), which is released in circulation during bacterial pneumonia in humans and displays intrinsic innate immune properties and cellular repair properties. DARS2 interacts with a bacterial-induced ubiquitin E3 ligase subunit, FBXO24, which targets the synthetase for ubiquitylation and degradation, a process that is inhibited by DARS2 acetylation. During experimental pneumonia, Fbxo24 knockout mice exhibit elevated DARS2 levels with an increase in pulmonary cellular and cytokine levels. In silico modeling identified an FBXO24 inhibitory compound with immunostimulatory properties which extended DARS2 lifespan in cells. Here, we show a unique biological role for an extracellular, mitochondrially derived enzyme and its molecular control by the ubiquitin apparatus, which may serve as a mechanistic platform to enhance protective host immunity through small molecule discovery. Here, Johnson et al show that bacterial pneumonia induces an interaction between the ubiquitin E3 ligase subunit FBX024 and the mitochondrial protein DARS2 to modulate host immune responses.

Background Fatty acid oxidation (FAO) defects have been implicated in experimental models of acute lung injury and associated with poor outcomes in critical illness. In this study, we examined acylcarnitine profiles and 3-methylhistidine as markers of FAO defects and skeletal muscle catabolism, respectively, in patients with acute respiratory failure. We determined whether these metabolites were associated with host-response ARDS subphenotypes, inflammatory biomarkers, and clinical outcomes in acute respiratory failure. Methods In a nested case–control cohort study, we performed targeted analysis of serum metabolites of patients intubated for airway protection (airway controls), Class 1 (hypoinflammatory), and Class 2 (hyperinflammatory) ARDS patients (N = 50 per group) during early initiation of mechanical ventilation. Relative amounts were quantified by liquid chromatography high resolution mass spectrometry using isotope-labeled standards and analyzed with plasma biomarkers and clinical data. Results Of the acylcarnitines analyzed, octanoylcarnitine levels were twofold increased in Class 2 ARDS relative to Class 1 ARDS or airway controls ( P  = 0.0004 and < 0.0001, respectively) and was positively associated with Class 2 by quantile g-computation analysis ( P  = 0.004). In addition, acetylcarnitine and 3-methylhistidine were increased in Class 2 relative to Class 1 and positively correlated with inflammatory biomarkers. In all patients within the study with acute respiratory failure, increased 3-methylhistidine was observed in non-survivors at 30 days ( P  = 0.0018), while octanoylcarnitine was increased in patients requiring vasopressor support but not in non-survivors ( P  = 0.0001 and P  = 0.28, respectively). Conclusions This study demonstrates that increased levels of acetylcarnitine, octanoylcarnitine, and 3-methylhistidine distinguish Class 2 from Class 1 ARDS patients and airway controls. Octanoylcarnitine and 3-methylhistidine were associated with poor outcomes in patients with acute respiratory failure across the cohort independent of etiology or host-response subphenotype. These findings suggest a role for serum metabolites as biomarkers in ARDS and poor outcomes in critically ill patients early in the clinical course.

Eosinophilic inflammation is implicated in asthma. Eotaxin 1-3 regulate eosinophil trafficking into the airways along with other chemotactic factors. However, the epithelial and bronchoalveolar lavage (BAL) cell expression of these chemokines in relation to asthma severity and eosinophilic phenotypes has not been addressed. To measure the expression of the three eotaxin isoforms in bronchoscopically obtained samples and compare them with clinically relevant parameters between normal subjects and patients with asthma. Normal subjects and patients with asthma of varying severity recruited through the Severe Asthma Research Program underwent clinical assessment and bronchoscopy with airway brushing and BAL. Eotaxin 1-3 mRNA/protein were measured in epithelial and BAL cells and compared with asthma severity, control and eosinophilic inflammation. Eotaxin-2 and eotaxin-3 mRNA and eotaxin-2 protein were increased in airway epithelial brushings from patients with asthma and were highest in cases of severe asthma (p values 0.0155, 0.0033 and 0.0006, respectively), with eotaxin-2 protein increased with age at onset. BAL cells normally expressed high levels of eotaxin-2 mRNA/protein but BAL fluid levels of eotaxin-2 were lowest in severe asthma. Epithelial eotaxin-2 and eotaxin-3 mRNA/protein was associated with sputum eosinophilia, lower forced expiratory volume in 1 s and more asthma exacerbations. Airway epithelial cell eotaxin-2 protein differed by asthma severity only in those with late onset disease, and tended to be highest in those with late onset eosinophilic asthma. Epithelial eotaxin-2 and 3 are increased in asthma and severe asthma. Their expression may contribute to luminal migration of eosinophils, especially in later onset disease, asthma control and severity.

Idiopathic pulmonary fibrosis is a lethal parenchymal lung disease characterized by denudation of the lung epithelium, fibroblast proliferation, and collagen deposition. Cellular changes underlying disease progression involve injury to alveolar epithelial cells, epithelial to mesenchymal transition, proliferation of alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts and of fibroblasts resulting in enhanced deposition of extracellular matrix proteins. Hepatocyte growth factor (HGF) inhibits progression of bleomycin-induced pulmonary fibrosis in mice. The mechanism underlying the inhibitory effect of HGF was investigated in an in vitro model. We show that HGF markedly antagonizes basal and transforming growth factor (TGF)-beta-induced expression of myofibroblast markers such as alpha-SMA, collagen type 1, and fibronectin in rat alveolar epithelial cells. HGF also inhibited TGF-beta-induced alpha-SMA expression in primary murine alveolar epithelial cells. Since TGF-beta is known to regulate alpha-SMA expression, the effect of HGF on components of TGF-beta signaling was investigated. HGF induced expression of Smad7, an inhibitor of TGF-beta signaling, in a mitogen-activated protein kinase-dependent manner. HGF also induced the nuclear export of Smad7 and Smad ubiquitin regulatory factor 1 (Smurf1) to the cytoplasm. HGF-dependent decrease in alpha-SMA was abolished with specific siRNAs targeted to Smad7. Thus, induction of Smad7 by HGF serves to limit acquisition of the myofibroblast phenotype in alveolar epithelial cells.