Bacteriophage (phage) therapy, which uses lytic viruses as antimicrobials, is a potential strategy to address the antimicrobial resistance crisis. Cystic fibrosis, a disease complicated by recurrent Pseudomonas aeruginosa pulmonary infections, is an example of the clinical impact of antimicrobial resistance. Here, using a personalized phage therapy strategy that selects phages for a predicted evolutionary trade-off, nine adults with cystic fibrosis (eight women and one man) of median age 32 (range 22-46) years were treated with phages on a compassionate basis because their clinical course was complicated by multidrug-resistant or pan-drug-resistant Pseudomonas that was refractory to prior courses of standard antibiotics. The individuals received a nebulized cocktail or single-phage therapy without adverse events. Five to 18 days after phage therapy, sputum Pseudomonas decreased by a median of 104 CFU ml-1, or a mean difference of 102 CFU ml-1 (P = 0.006, two-way analysis of variance with Dunnett's multiple-comparisons test), without altering sputum microbiome, and an analysis of sputum Pseudomonas showed evidence of trade-offs that decreased antibiotic resistance or bacterial virulence. In addition, an improvement of 6% (median) and 8% (mean) predicted FEV1 was observed 21-35 days after phage therapy (P = 0.004, Wilcoxon signed-rank t-test), which may reflect the combined effects of decreased bacterial sputum density and phage-driven trade-offs. These results show that a personalized, nebulized phage therapy trade-off strategy may affect clinical and microbiologic endpoints, which must be evaluated in larger clinical trials.

Axonemal dynein, the macromolecular machine that powers ciliary motility, assembles in the cytosol with the help of dynein axonemal assembly factors (DNAAFs). These DNAAFs localize in cytosolic foci thought to form via liquid-liquid phase separation. However, the functional significance of DNAAF foci formation and how the production and assembly of multiple components are so efficiently coordinated, at such enormous scale, remain unclear. Here, we unveil an axonemal dynein production and assembly hub enriched with translating heavy chains (HCs) and DNAAFs. We show that mRNAs encoding interacting HCs of outer dynein arms colocalize in cytosolic foci, along with nascent HCs. The formation of these mRNA foci and their colocalization relies on HC translation. We observe that a previously identified DNAAF assembly, containing the DNAAF Lrrc6 and cochaperones Ruvbl1 and Ruvbl2, colocalizes with these HC foci, and is also dependent on HC translation. We additionally show that Ruvbl1 is required for the recruitment of Lrrc6 into the HC foci and that both proteins function cotranslationally. We propose that these DNAAF foci are anchored by stable interactions between translating HCs, ribosomes, and encoding mRNAs, followed by cotranslational molecular condensation of cochaperones and assembly factors, providing a potential mechanism that coordinates HC translation, folding, and assembly at scale.

Background and PurposeFibrotic lung remodelling after a respiratory viral infection represents a debilitating clinical sequela. Studying or managing viral-fibrotic sequela remains challenging, due to limited therapeutic options and lack of understanding of mechanisms. This study determined whether protein disulfide isomerase A3 (PDIA3) and secreted phosphoprotein 1 (SPP1), which are associated with pulmonary fibrosis, can promote influenza-induced lung fibrotic remodelling and whether inhibition of PDIA3 or SPP1 can resolve viral-mediated fibrotic remodelling.Experimental ApproachA retrospective analysis of TriNetX data sets was conducted. Serum from healthy controls and influenza A virus (IAV)-infected patients was analysed. An inhibitor of PDIA3, punicalagin, and a neutralizing antibody for SPP1 were administered in mice. Macrophage cells treated with macrophage colony-stimulating factor (M-CSF) were used as a cell culture model.Key ResultsThe TriNetX data set showed an increase in lung fibrosis and decline in lung function in flu-infected acute respiratory distress syndrome (ARDS) patients compared with non-ARDS patients. Serum samples revealed a significant increase in SPP1 and PDIA3 in influenza-infected patients. Lung PDIA3 and SPP1 expression increased following viral infection in mouse models. Punicalagin administration 2 weeks after IAV infection in mice caused a significant decrease in lung fibrosis and improved oxygen saturation. Administration of neutralizing SPP1 antibody decreased lung fibrosis. Inhibition of PDIA3 decreased SPP1secretion from macrophages, in association with diminished disulfide bonds in SPP1.Conclusion and ImplicationsThe PDIA3-SPP1 axis promotes post-influenza lung fibrosis in mice and that pharmacological inhibition of PDIA3 or SPP1 can treat virus-induced lung fibrotic sequela. image

Sputum induction (SI) is a non-invasive technique to obtain lower airway samples from people who do not spontaneously produce sputum. Since its introduction in 1958, its use has significantly increased for various respiratory conditions, especially with the establishment of standardized techniques. It has been used in pediatric research since 2002, starting with asthma studies. It is a safe and repeatable procedure for non-expectorating children. The ability to analyze both cellular and fluid components of sputum provides valuable information about lung diseases. This work reviews the utility of sputum induction in pediatric respiratory research and its potential clinical applications. We also discuss the safety, tolerability, and side effects profile of the procedure, emphasizing its significance in pediatric respiratory research. Despite the valuable insights gained from SI studies, challenges remain for widespread clinical utilization, requiring further refinement of the technique and increased accessibility in clinical settings.

Secondary lung infection by inhaled Staphylococcus aureus (SA) is a common and lethal event for individuals infected with influenza A virus (IAV). How IAV disrupts host defense to promote SA infection in lung alveoli, where fatal lung injury occurs, is not known. We addressed this issue using real-time determinations of alveolar responses to IAV in live, intact, perfused lungs. Our findings show that IAV infection blocked defensive alveolar wall liquid (AWL) secretion and induced airspace liquid absorption, thereby reversing normal alveolar liquid dynamics and inhibiting alveolar clearance of inhaled SA. Loss of AWL secretion resulted from inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel in the alveolar epithelium, and airspace liquid absorption was caused by stimulation of the alveolar epithelial Na+ channel (ENaC). Loss of AWL secretion promoted alveolar stabilization of inhaled SA, but rescue of AWL secretion protected against alveolar SA stabilization and fatal SA-induced lung injury in IAV-infected mice. These findings reveal a central role for AWL secretion in alveolar defense against inhaled SA and identify AWL inhibition as a critical mechanism of IAV lung pathogenesis. AWL rescue may represent a new therapeutic approach for IAV-SA coinfection.

Cystic fibrosis is a life-shortening genetic disorder, caused by mutations in the gene that encodes cystic fibrosis transmembrane-conductance regulator, a cAMP-activated chloride and bicarbonate channel. Persistent neutrophilic inflammation is a major contributor to cystic fibrosis lung disease. However, how cystic fibrosis transmembrane-conductance regulator loss of function leads to excessive inflammation and its clinical sequela remains incompletely understood. In this study, neutrophils from F508del-CF and healthy control participants were compared for gene transcription. We found that cystic fibrosis circulating neutrophils have a prematurely primed basal state with significantly higher scores for activation, chemotaxis, immune signaling, and pattern recognition. Such an irregular basal state appeared not related to the blood environment and was also observed in neutrophils derived from the F508del-CF HL-60 cell line, indicating an innate characteristic of the phenotype. Lipopolysaccharides (LPS) stimulation drastically shifted the transcriptional landscape of healthy control neutrophils toward a robust immune response; however, cystic fibrosis neutrophils were immune-exhausted, reflected by abnormal cell aging and fate determination in gene programming. Moreover, cystic fibrosis sputum neutrophils differed significantly from cystic fibrosis circulating neutrophils in gene transcription with increased inflammatory response, aging, apoptosis, and necrosis, suggesting additional environmental influences on the neutrophils in cystic fibrosis lungs. Taken together, our data indicate that loss of cystic fibrosis transmembrane-conductance regulator function has intrinsic effects on neutrophil immune programming, leading to premature priming and dysregulated response to challenge.

As we characterize the clinical benefits of highly effective modulator therapy (HEMT) in the cystic fibrosis (CF) population, our paradigm for treating and monitoring disease continues to evolve. More sensitive approaches are necessary to detect early disease and clinical progression. This article reviews evolving strategies to assess disease control and progression in the HEMT era. This article also explores developments in pulmonary function monitoring, advanced respiratory imaging, tools for the collection of patient-reported outcomes, and their application to profile individual responses, guide therapeutic decisions, and improve the quality of life of people with CF.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide, however our understanding of cell specific mechanisms underlying COPD pathobiology remains incomplete. Here, we analyze single-cell RNA sequencing profiles of explanted lung tissue from subjects with advanced COPD or control lungs, and we validate findings using single-cell RNA sequencing of lungs from mice exposed to 10 months of cigarette smoke, RNA sequencing of isolated human alveolar epithelial cells, functional in vitro models, and in situ hybridization and immunostaining of human lung tissue samples. We identify a subpopulation of alveolar epithelial type II cells with transcriptional evidence for aberrant cellular metabolism and reduced cellular stress tolerance in COPD. Using transcriptomic network analyses, we predict capillary endothelial cells are inflamed in COPD, particularly through increased CXCL-motif chemokine signaling. Finally, we detect a high-metallothionein expressing macrophage subpopulation enriched in advanced COPD. Collectively, these findings highlight cell-specific mechanisms involved in the pathobiology of advanced COPD.

The development of the porcine model of cystic fibrosis (CF) disease marked a leap forward in our ability to understand CF pathogenesis while keeping a high degree of fidelity with human disease (1, 2). Similarly, the emergence of single-cell RNA sequencing (scRNAseq) as a tool for gene expression analysis rapidly expanded our CF-specific transcriptome knowledge with unprecedented resolution (3, 4). Early studies of scRNAseq in airway epithelial and immune cells provided data on dysfunctional transcriptional programs controlling ion transport, host defense, and immunity within CF airways and even enabled the discovery of novel airway epithelial cell types (4–6). In this issue of the Journal, Thurman and colleagues (pp. 612–622) integrate these groundbreaking developments to chart the transcriptional topography of the porcine bronchial tree at birth, providing for the first time a detailed landscape of gene expression and epithelial cell–type distribution in newborn CFTR (CF transmembrane conductance regulator)–deficient (CFTR) lungs (7). Through this work, the authors inch ever closer to answering a long-standing question: does CF disease originate from intrinsic epithelial dysfunction, or is epithelial dysfunction a result of cyclical infection and inflammation in the postnatal airway environment? The authors microdissected large and small airways from newborn CFTR pigs for scRNAseq analysis. Despite CFTR deficiency, the transcriptome of CF and non-CF porcine airways exhibited minimal transcriptomic differences at birth, suggesting that CFTR deficiency itself did not cause extensive epithelial gene expression dysregulation. This remarkable finding brings forth the idea that CF lung disease may be a modifiable, largely postnatal process rather than a predetermined outcome due to intrinsic epithelial dysfunction and inflammation already evolving in utero. Although minimal transcriptional differences were observed between CF and non-CF airways, the authors show vastly different transcriptomic and protein marker profiles between small and large airways in CFTR and wild-type porcine lungs. Apart from CFTR, a considerable number of differentially expressed genes distinguished basal, secretory, and ciliated cells from large and small airways, with remarkably fewer differentially expressed genes in “deeper” cells (e.g., muscle cells, endothelial cells) than in those closer to the apical surface (e.g., ciliated cells, secretory cells). Although others have characterized epithelial and immune cell subgroups in human CF and experimental models using scRNAseq, this work offers a detailed map of the airway epithelium to understand cell subset distributions and transcriptional topography in the earliest stages of CF. This work also provides a point of reference to investigate CF pathogenesis, from relatively unaffected airways to the chronic infection and inflammation in our closest animal model of CF lung disease to date. This may be particularly relevant to the role of small airways, an incompletely characterized, yet often proposed site of early CF lung disease development (8–10). This study confirms the presence of common airway epithelial cells in small and large airways, including ciliated, secretory, and club cells. It also identifies rare epithelial cells such as the recently described ionocytes in a nearly exclusive large airway distribution (4). The paucity of ionocytes in small airways concurred with increased expression of non-CFTR ion channels in small airway epithelia, highlighting howmuch remains to be understood about the function of these cells and their transcriptional and functional equivalents in smaller airways. Ion transport is critically disrupted in CF, leading to abnormal solute concentration in respiratory secretions, mucus desiccation, and impaired mucociliary clearance (8, 11, 12). Expression of CFTR, a major driver of epithelial chloride, bicarbonate, and fluid transport, was overall low in wild-type airways. It was measured at the highest concentration in ionocytes, followed by secretory, ciliated, and basal cells. Regionally, CFTR expression was highest in ionocytes of large airways and in secretory and ciliated cells of small airways. Small airways expressed more epithelial sodium channel subunits, less of its negative regulator, BPIFA1 (bactericidal/permeability-increasing fold containing family A, member 1) (13), and more aquaporins than large airways, suggesting a program that enhances sodiummovement and osmotic water transport in smaller airways. Functionally, large and small airways had similar paracellular conductance, but transcriptional differences suggest that small airways were more permeable to cations than anions, whereas large ones had similar permeability to both. These findings will require further functional validation. Mucins are crucial contributors to the clinical manifestations of CF, and their expression is influenced by inflammation and epithelial dysfunction (14, 15). The authors found that MUC5AC (mucin 5AC, oligomeric mucus/gel-forming) andMUC5B (mucin 5B, oligomeric mucus/gel-forming) expression was site specific, with a transition fromMUC5AC-rich secretory cells to MUC5AC-low club cells distally in small airways. These observations suggested a site-specific transcriptional program that promotes higher mucin content in large airways. These initial observations provide a reference to investigate epithelial cell and mucin production by lower airways that may foment chronic inflammation and promote mucoid impaction. The secretion of host defense proteins and peptides (AMPs) is a key component of epithelial immunity (16, 17). In neonatal CF porcine lungs, secretory cells expressed the most AMPs, again with remarkable regional differences: large airways expressed higher concentrations of lysozyme, lactoferrin, calprotectin, and BPIFA1 (a surfactant AMP with ion transport and immunomodulatory properties) (18). In contrast, the small airway epithelium differentially

Persistent neutrophil-dominated lung inflammation contributes to lung damage in cystic fibrosis (CF). However, the mechanisms that drive persistent lung neutrophilia and tissue deterioration in CF are not well characterized. Starting from the observation that, in patients with CF, c-c motif chemokine receptor 2 (CCR2)+ monocytes/macrophages are abundant in the lungs, we investigate the interplay between monocytes/macrophages and neutrophils in perpetuating lung tissue damage in CF. Here we show that CCR2+ monocytes in murine CF lungs drive pathogenic transforming growth factor β (TGF-β) signaling and sustain a pro-inflammatory environment by facilitating neutrophil recruitment. Targeting CCR2 to lower the numbers of monocytes in CF lungs ameliorates neutrophil inflammation and pathogenic TGF-β signaling and prevents lung tissue damage. This study identifies CCR2+ monocytes as a neglected contributor to the pathogenesis of CF lung disease and as a therapeutic target for patients with CF, for whom lung hyperinflammation and tissue damage remain an issue despite recent advances in CF transmembrane conductance regulator (CFTR)-specific therapeutic agents.

TOPIC: Genetic and Developmental Disorders TYPE: Original Investigations PURPOSE: Cystic Fibrosis (CF) is a multisystem autosomal recessive disease caused by mutations in the CFTR (CF transmembrane conductance regulator) gene. Airway inflammation and infection in CF leads to tissue injury, worsening lung function, and disease progression. Increased cytokine levels reflect the exaggerated inflammation characteristic of the CF airway environment and contribute to pathogenesis even in the absence of respiratory symptoms. We sought to define individual CF-specific cytokine variations as a means to characterize the difference between healthy and CF airways, and to identify patients at risk of clinical deterioration. METHODS: Sputum samples were obtained from Forty-four adult patients with confirmed CF diagnosis from the Yale Adult CF Program. Patients were classified as CF stable at each visit if they did not meet predefined criteria for an acute exacerbation. Ten healthy controls (HC) were recruited to undergo sputum induction. Sputum samples were assayed for twelve cytokines using a multiplexed indirect ELISA. Differences in cytokine levels between CF stable and healthy controls were analyzed using the Mann-Whitney Test with Bonferroni correction. RESULTS: Twelve inflammatory cytokine levels were measured. Six cytokines were significantly increased in the sputum of stable CF patients compared to healthy controls (median values, pg/mL). These cytokines included: IL-1beta (CF 1720; HC 39.7), IL-8 (CF 28173; HC 3468), TNF-alpha (CF 98.65; HC 10.70), and IFN-alpha (CF 38.90; HC 5.60) [p < 0.0001] as well as IFN-gamma (CF 68.45; HC 17.10) and IL-13 (20.10; 0.00) [p < 0.01]. Cytokines tested without significant difference between groups included: CXCL10, G-CSF, IFNI, IL-6, MCP1, and MIP-alpha. CONCLUSIONS: Inflammatory cytokine levels of IL-1beta, IL-8, TNF-alpha, IFN-a, IFN-gamma and IL-13 are significantly increased in the sputum of stable CF patients compared to the sputum of healthy controls. CLINICAL IMPLICATIONS: Monitoring basal sputum cytokine levels may help identify patients with worsening inflammation before symptoms develop and thus prevent irreversible lung damage and disease progression. Additionally, further understanding of inflammatory cytokine dysregulation in stable CF airways may serve as the basis for a sputum inflammation marker panel that guides future therapeutic interventions. DISCLOSURES: No relevant relationships by Clemente Britto, source=Web Response Consultant relationship with AstraZeneca Please note: >$100000 by Geoffrey Chupp, source=Web Response, value=Consulting fee Consultant relationship with Genentech Please note: $20001 - $100000 by Geoffrey Chupp, source=Web Response, value=Consulting fee Consultant relationship with Genzyme Corporation Please note: $5001 - $20000 by Geoffrey Chupp, source=Web Response, value=Consulting fee Consultant relationship with Novartis Please note: $5001 - $20000 by Geoffrey Chupp, source=Web Response, value=Consulting fee Consultant relationship with Circassia Please note: $1001 - $5000 by Geoffrey Chupp, source=Web Response, value=Consulting fee Consultant relationship with Boehringer Ingelheim Please note: $1001 - $5000 by Geoffrey Chupp, source=Web Response, value=Consulting fee Consultant relationship with GlaxoSmithKline Please note: $1-$1000 by Geoffrey Chupp, source=Web Response, value=Consulting fee Consultant relationship with Regeneron Please note: 5/2019-present Added 04/26/2021 by Lauren Cohn, source=Web Response, value=Consulting fee Speaker/Speaker's Bureau relationship with Genentech Please note: 5/2019-11/2019 Added 04/26/2021 by Lauren Cohn, source=Web Response, value=Honoraria Advisory Committee Member relationship with novartis Please note: 10/2019 Added 04/26/2021 by Lauren Cohn, source=Web Response, value=Consulting fee Advisory Committee Member relationship with glaxosmithkline Please note: 12/2019 Added 04/26/2021 by Lauren Cohn, source=Web Response, value=Consulting fee Advisory Committee Member relationship with sanofi Please note: 5/2020 Added 04/26/2021 by Lauren Cohn, source=Web Response, value=Consulting fee Advisory Committee Member relationship with Pieris Please note: 5/2019 Added 04/26/2021 by Lauren Cohn, source=Web Response, value=Consulting fee Advisory Committee Member relationship with AstraZeneca Please note: 5/2019-7/2020 Added 04/26/2021 by Lauren Cohn, source=Web Response, value=Consulting fee Consultant relationship with Biohaven Please note: 6/23/2020 Added 04/26/2021 by Lauren Cohn, source=Web Response, value=Consulting fee No relevant relationships by Sara Khanal, source=Web Response No relevant relationships by qing liu, source=Web Response No relevant relationships by Jana Zielonka, source=Web Response

BackgroundAsthma has been associated with impaired interferon response. Multiple cell types have been implicated in such response impairment and may be responsible for asthma immunopathology. However, existing models to study the immune response in asthma are limited by bulk profiling of cells. Our objective was to Characterize a model of peripheral blood mononuclear cells (PBMCs) of patients with severe asthma (SA) and its response to the TLR3 agonist Poly I:C using two single-cell methods.MethodsTwo complementary single-cell methods, DropSeq for single-cell RNA sequencing (scRNA-Seq) and mass cytometry (CyTOF), were used to profile PBMCs of SA patients and healthy controls (HC). Poly I:C-stimulated and unstimulated cells were analyzed in this study.ResultsPBMCs (n=9414) from five SA (n=6099) and three HC (n=3315) were profiled using scRNA-Seq. Six main cell subsets, namely CD4+T cells, CD8+T cells, natural killer (NK) cells, B cells, dendritic cells (DCs), and monocytes, were identified. CD4+T cells were the main cell type in SA and demonstrated a pro-inflammatory profile characterized by increased JAK1 expression. Following Poly I:C stimulation, PBMCs from SA had a robust induction of interferon pathways compared with HC. CyTOF profiling of Poly I:C stimulated and unstimulated PBMCs (n=160,000) from the same individuals (SA=5; HC=3) demonstrated higher CD8+and CD8+effector T cells in SA at baseline, followed by a decrease of CD8+effector T cells after poly I:C stimulation.ConclusionsSingle-cell profiling of an in vitro model using PBMCs in patients with SA identified activation of pro-inflammatory pathways at baseline and strong response to Poly I:C, as well as quantitative changes in CD8+effector cells. Thus, transcriptomic and cell quantitative changes are associated with immune cell heterogeneity in this model to evaluate interferon responses in severe asthma.

The pathogenesis of chronic obstructive pulmonary disease (COPD) involves aberrant responses to cellular stress caused by chronic cigarette smoke (CS) exposure. However, not all smokers develop COPD and the critical mechanisms that regulate cellular stress responses to increase COPD susceptibility are not understood. Because microRNAs are well-known regulators of cellular stress responses, we evaluated microRNA expression arrays performed on distal parenchymal lung tissue samples from 172 subjects with and without COPD. We identified miR-24-3p as the microRNA that best correlated with radiographic emphysema and validated this finding in multiple cohorts. In a CS exposure mouse model, inhibition of miR-24-3p increased susceptibility to apoptosis, including alveolar type II epithelial cell apoptosis, and emphysema severity. In lung epithelial cells, miR-24-3p suppressed apoptosis through the BH3-only protein BIM and suppressed homology-directed DNA repair and the DNA repair protein BRCA1. Finally, we found BIM and BRCA1 were increased in COPD lung tissue, and BIM and BRCA1 expression inversely correlated with miR-24-3p. We concluded that miR-24-3p, a regulator of the cellular response to DNA damage, is decreased in COPD, and decreased miR-24-3p increases susceptibility to emphysema through increased BIM and apoptosis.

Acute pulmonary Exacerbations (AE) are episodes of clinical worsening in cystic fibrosis (CF), often precipitated by infection. Timely detection is critical to minimize the morbidity and lung function decline associated with acute inflammation during AE. We previously demonstrated that the airway protein Short Palate Lung Nasal epithelium Clone 1 (SPLUNC1) is regulated by inflammatory signals. Here, we investigated the use of SPLUNC1 fluctuations to diagnose and predict AE in CF. We enrolled adult CF subjects from two independent cohorts to measure AE markers of inflammation in sputum and recorded clinical outcomes for a 1-year follow-up period. SPLUNC1 levels were high in healthy control sputum (n=9, 10.7µg/mL), and significantly decreased in CF subjects without AE (n=30, 5.7µg/mL, p=0.016). SPLUNC1 levels were 71.9% lower during AE (n=14, 1.6µg/mL, p=0.0034) regardless of age, sex, CF-causing mutation, or microbiology findings. Cytokines Il-1β and TNFα were also increased in AE, whereas lung function did not consistently decrease. Stable CF subjects with lower SPLUNC1 levels were much more likely to have an AE at 60 days (Hazard Ratio: 11.49, Standard Error: 0.83, p=0.0033). Low-SPLUNC1 stable subjects remained at higher AE risk even one year after sputum collection (Hazard Ratio: 3.21, Standard Error: 0.47, p=0.0125). SPLUNC1 was transcriptionally downregulated by inflammatory cytokines and degraded by proteases increased in sputum during AE. Our findings suggest that low sputum SPLUNC1 levels could detect subjects at increased risk of AE in order to guide early therapeutic interventions in CF.

Staphylococcus aureus is a prominent human pathogen that readily adapts to host immune defenses. Here, we show that, in contrast to Gram-negative pathogens, S. aureus induces a distinct airway immunometabolic response dominated by the release of the electrophilic metabolite, itaconate. The itaconate synthetic enzyme, IRG1, is activated by host mitochondrial stress, which is induced by staphylococcal glycolysis. Itaconate inhibits S. aureus glycolysis and selects for strains that re-direct carbon flux to fuel extracellular polysaccharide (EPS) synthesis and biofilm formation. Itaconate-adapted strains, as illustrated by S. aureus isolates from chronic airway infection, exhibit decreased glycolytic activity, high EPS production, and proficient biofilm formation even before itaconate stimulation. S. aureus thus adapts to the itaconate-dominated immunometabolic response by producing biofilms, which are associated with chronic infection of the human airway.

It has been postulated that the underlying pathophysiology of COVID-19 is mediated by cytokine storm resulting in a hyperinflammatory state. A similar kind of cytokine-storm has been described in individuals undergoing veno-venous extracorporeal membrane oxygenation (VV ECMO) support. There is therefore concern that initiation of VV ECMO support among COVID19 patients could further exacerbate this dysregulated inflammatory response. In this prospective cohort study, we describe the clinical course and cytokine fluctuations in eight subjects treated with VV ECMO for management of refractory respiratory failure from COVID19. Among all eight patients, cytokine elevations were noted among Interleukin 6 (IL-6), Interleukin 10 (IL-10), and Interleukin 2 Receptor (CD25) soluble (sIL2R). Although further research is necessary, among our cohort of patients it did not appear that initiation of VV ECMO worsened cytokine storm.

Chronic Obstructive Pulmonary Disease (COPD) is a leading cause of death worldwide. To identify cell-specific mechanisms underlying COPD pathobiology, we analysed single-cell RNA sequencing (scRNAseq) profiles of explanted lung tissue from subjects with advanced COPD or control lungs. Findings were validated with scRNAseq of lungs from mice exposed to 10 months of cigarette smoke (CS), isolated human alveolar epithelial cells, and immunostaining of human lung tissue samples. We identified a subpopulation of alveolar epithelial type II cells with transcriptional evidence for aberrant cellular metabolism and reduced cellular stress tolerance, exemplified by decreased expression of the stress-response gene NUPR1. Network analyses identified an important role for inflamed capillary endothelial cells in COPD, particularly through CXCL-motif chemokine signalling. Finally, we detected a metallothionein expressing macrophage subpopulation unique to COPD. Collectively, these findings highlight cell-specific mechanisms involved in the pathobiology of advanced COPD.

The bacterium Pseudomonas aeruginosa is especially pathogenic, often being associated with intractable pneumonia and high mortality. How P. aeruginosa avoids immune clearance and persists in the inflamed human airway remains poorly understood. In this study, we show that P. aeruginosa can exploit the host immune response to maintain infection. Notably, unlike other opportunistic bacteria, we found that P. aeruginosa alters its metabolic and immunostimulatory properties in response to itaconate, an abundant host-derived immunometabolite in the infected lung. Itaconate induces bacterial membrane stress, resulting in downregulation of lipopolysaccharides (LPS) and upregulation of extracellular polysaccharides (EPS). These itaconate-adapted P. aeruginosa accumulate lptD mutations, which favor itaconate assimilation and biofilm formation. EPS, in turn, induces itaconate production by myeloid cells, both in the airway and systemically, skewing the host immune response to one permissive of chronic infection. Thus, the metabolic versatility of P. aeruginosa needs to be taken into account when designing therapies.