Here, we describe a novel pathogenic entity, the activated PMN (polymorphonuclear leukocyte, i.e., neutrophil)-derived exosome. These CD63 + /CD66b + nanovesicles acquire surface-bound neutrophil elastase (NE) during PMN degranulation, NE being oriented in a configuration resistant to a1-antitrypsin (a1AT). These exosomes bind and degrade extracellular matrix (ECM) via the integrin Mac-1 and NE, respectively, causing the hallmarks of chronic obstructive pulmonary disease (COPD). Due to both ECM targeting and a1AT resistance, exosomal NE is far more potent than free NE. Importantly, such PMN-derived exosomes exist in clinical specimens from subjects with COPD but not healthy controls and are capable of transferring a COPD-like phenotype from humans to mice in an NE-driven manner. Similar findings were observed for another neutrophil-driven disease of ECM remodeling (bronchopulmonary dysplasia [BPD]). These findings reveal an unappreciated role for exosomes in the pathogenesis of disorders of ECM homeostasis such as COPD and BPD, providing a critical mechanism for proteolytic damage.

Rationale: Neutrophils are recruited to the airways of individuals with cystic fibrosis (CF). In adolescents and adults with CF, airway neutrophils actively exocytose the primary granule protease elastase (NE), whose extracellular activity correlates with lung damage. During childhood, free extracellular NE activity is measurable only in a subset of patients, and the exocytic function of airway neutrophils is unknown.Objectives: To measure NE exocytosis by airway neutrophils in relation to free extracellular NE activity and lung damage in children with CF.Methods: We measured lung damage using chest computed tomography coupled with the Perth-Rotterdam Annotated Grid Morphometric Analysis for Cystic Fibrosis scoring system. Concomitantly, we phenotyped blood and BAL fluid leukocytes by flow and image cytometry, and measured free extracellular NE activity using spectrophotometric and Förster resonance energy transfer assays. Children with airway inflammation linked to aerodigestive disorder were enrolled as control subjects.Measurements and Main Results: Children with CF but not disease control children harbored BAL fluid neutrophils with high exocytosis of primary granules, before the detection of bronchiectasis. This measure of NE exocytosis correlated with lung damage (R = 0.55; P = 0.0008), whereas the molecular measure of free extracellular NE activity did not. This discrepancy may be caused by the inhibition of extracellular NE by BAL fluid antiproteases and its binding to leukocytes.Conclusions: NE exocytosis by airway neutrophils occurs in all children with CF, and its cellular measure correlates with early lung damage. These findings implicate live airway neutrophils in early CF pathogenesis, which should instruct biomarker development and antiinflammatory therapy in children with CF.

Neutrophils are recruited to the airways of patients with acute respiratory distress syndrome (ARDS) where they acquire an activated pro-survival phenotype with an enhanced respiratory burst thought to contribute to ARDS pathophysiology. Our in vitro model enables blood neutrophil transepithelial migration into cell-free tracheal aspirate fluid from patients to recapitulate the primary airway neutrophil phenotype observed in vivo . Neutrophils transmigrated through our model toward airway fluid from children with lower respiratory viral infections coinfected with bacteria had elevated levels of neutrophil activation markers but paradoxically exhibited an inability to kill bacteria and a defective respiratory burst compared with children without bacterial coinfection. The airway fluid from children with bacterial coinfections had higher levels of neutrophil elastase activity, as well as myeloperoxidase levels compared to children without bacterial coinfection. Neutrophils transmigrated into the aspirate fluid from children with bacterial coinfection showed decreased respiratory burst and killing activity against H . influenzae and S . aureus compared to those transmigrated into the aspirate fluid from children without bacterial coinfection. Use of a novel transmigration model recapitulates this pathological phenotype in vitro that would otherwise be impossible in a patient, opening avenues for future mechanistic and therapeutic research.

Neutrophils have a prominent role in all human immune responses against any type of pathogen or stimulus. The lungs are a major neutrophil reservoir and neutrophilic inflammation is a primary response to both infectious and non-infectious challenges. While neutrophils are well known for their essential role in clearance of bacteria, they are also equipped with specific mechanisms to counter viruses and fungi. When these defense mechanisms become aberrantly activated in the absence of infection, this commonly results in debilitating chronic lung inflammation. Clearance of bacteria by phagocytosis is the hallmark role of neutrophils and has been studied extensively. New studies on neutrophil biology have revealed that this leukocyte subset is highly adaptable and fulfills diverse roles. Of special interest is how these adaptations can impact the outcome of an immune response in the lungs due to their potent capacity for clearing infection and causing damage to host tissue. The adaptability of neutrophils and their propensity to influence the outcome of immune responses implicates them as a much-needed target of future immunomodulatory therapies. This review highlights the recent advances elucidating the mechanisms of neutrophilic inflammation, with a focus on the lung environment due to the immense and growing public health burden of chronic lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), and acute lung inflammatory diseases such as transfusion-related acute lung injury (TRALI).

Highlights d Neutrophils recruited to cystic fibrosis airways undergo de novo RNA transcription d Neutrophil adaptation to CF airways leads to downregulation of their antimicrobial genes d Transcriptional blockade restores CF airway neutrophil bactericidal capacity

IntroductionThe pathological course of several chronic inflammatory diseases, including cystic fibrosis, chronic obstructive pulmonary disease, and rheumatoid arthritis, features an aberrant innate immune response dominated by neutrophils. In cystic fibrosis, neutrophil burden and activity of neutrophil elastase in the extracellular fluid have been identified as strong predictors of lung disease severity.ReviewAlthough neutrophils are generally considered to be rigid, pre-programmed effector leukocytes, recent studies suggest extensive plasticity in how neutrophil functions unfold upon recruitment to peripheral tissues, and how they choose their ultimate fate. Indeed, upon migration to cystic fibrosis airways, neutrophils display dysregulated lifespan, metabolic activation, and altered effector and regulatory functions, consistent with profound adaptation and phenotypic reprogramming. Licensed by signals present in cystic fibrosis airway microenvironment to survive and develop these novel functions, neutrophils orchestrate, in partnership with the epithelium and with the resident microbiota, the evolution of a pathological microenvironment. This microenvironment is defined by altered proteolytic, redox, and metabolic balance and the presence of stable luminal structures in which neutrophils and microbes coexist.ConclusionsThe elucidation of molecular mechanisms driving neutrophil plasticity in vivo will open new treatment opportunities designed to modulate, rather than block, the crucial adaptive functions fulfilled by neutrophils. This review aims to outline emerging mechanisms of neutrophil plasticity and their participation in the building of pathological microenvironments in the context of cystic fibrosis and other diseases with similar features.

Highlights d SARS-CoV-2 and H1N1-induced ARDS show unique transcriptional signatures d Tissue remodeling pathways dominate the transcriptional profile of SARS-CoV-2 ARDS d SARS-CoV-2/H1N1 double infection resembles the lung antiviral response to H1N1

Cystic fibrosis (CF) lung disease progressively worsens from infancy to adulthood. Disease-driven changes in early CF airway fluid metabolites may identify therapeutic targets to curb progression.CF patients aged 12-38 months (n=24; three out of 24 later denoted as CF screen positive, inconclusive diagnosis) received chest computed tomography scans, scored by the Perth-Rotterdam Annotated Grid Morphometric Analysis for CF (PRAGMA-CF) method to quantify total lung disease (PRAGMA-%Dis) and components such as bronchiectasis (PRAGMA-%Bx). Small molecules in bronchoalveolar lavage fluid (BALF) were measured with high-resolution accurate-mass metabolomics. Myeloperoxidase (MPO) was quantified by ELISA and activity assays.Increased PRAGMA-%Dis was driven by bronchiectasis and correlated with airway neutrophils. PRAGMA-%Dis correlated with 104 metabolomic features (p<0.05, q<0.25). The most significant annotated feature was methionine sulfoxide (MetO), a product of methionine oxidation by MPO-derived oxidants. We confirmed the identity of MetO in BALF and used reference calibration to confirm correlation with PRAGMA-%Dis (Spearman's ρ=0.582, p=0.0029), extending to bronchiectasis (PRAGMA-%Bx; ρ=0.698, p=1.5×10), airway neutrophils (ρ=0.569, p=0.0046) and BALF MPO (ρ=0.803, p=3.9×10).BALF MetO associates with structural lung damage, airway neutrophils and MPO in early CF. Further studies are needed to establish whether methionine oxidation directly contributes to early CF lung disease and explore potential therapeutic targets indicated by these findings.